Transfected t-cells and t-cell receptors for use in immunotherapy against cancers

ABSTRACT

Disclosed are T-cell receptors (TCRs) binding to tumor-associated antigens (TAAs) for targeting cancer cells, T-cells expressing same, methods for producing same, and methods for treating cancers using same. Disclosed are TCRs and their variants that bind to HLA class I or II molecules with a peptide, such as MAG-003 have the amino acid sequence of KVLEHVVRV (SEQ ID NO:1). The description further relates to peptides, proteins, nucleic acids, cells for use in immunotherapeutic methods, the immunotherapy of cancer, and tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T-cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of U.S. patentapplication Ser. No. 16/403,058, filed May 3, 2019, which is aContinuation application of U.S. patent application Ser. No. 15/460,654,filed Mar. 16, 2017, now U.S. Pat. No. 10,538,573, issued Jan. 21, 2020,which claims the benefit of U.S. Provisional Application Ser. No.62/308,975, filed 16 Mar. 2016, and Great Britain Application No.1604492.7, filed 16 Mar. 2016, the content of each of these applicationsis herein incorporated by reference in their entirety.

This application also is related to PCT/EP2017/056260 filed 16 Mar.2017, the content of which is incorporated herein by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “3000058-002003_Sequence_Listing_ST25.txt” createdon 21 Jan. 2020, and 49,233 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

BACKGROUND

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). These tumorassociated antigens (TAAs) can be peptides derived from all proteinclasses, such as enzymes, receptors, transcription factors, etc. whichare expressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

MAGEA4 is a member of the MAGEA gene family. Expression of the MAGEA4protein and mRNA has been linked to the development and prognosis ofvarious cancers. MAG-003 peptide, i.e., KVLEHVVRV (SEQ ID NO:1), is anHLA-A*0201-restricted cytotoxic T lymphocyte (CTL) epitope of MAGEA4(amino acids 286-294). (Jia et al. 2010; Wu et al. 2011), the contentsof which are hereby incorporated by reference in their entirety. MAG-003elicits peptide-specific CTLs both in vitro from HLA-A*0201-positivePBMCs and in HLA-A*0201/Kb transgenic mice. MAG-003-induced CTLs lysetarget-cells in an HLA-A*0201-restricted fashion, demonstrating thatMAG-003 is an HLA-A*0201-restricted CTL epitope.

There are two classes of MHC-molecules, MHC class I and MHC class II.Complexes of peptide and MHC class I are recognized by CD8-positiveT-cells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T-cells bearing the appropriate TCR. Since bothtypes of response, CD8 and CD4 dependent, contribute jointly andsynergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens and corresponding T cellreceptors is important in the development of cancer immunotherapies suchas vaccines and cell therapies.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T-cells bearingspecific T-cell receptors (TCR). Therefore, TAAs are a starting pointfor the development of a T-cell based therapy including but not limitedto tumor vaccines and cell therapies.

In the case of targeting peptide-MHC by specific TCRs (e.g., solubleTCRs or cell-surface TCRs) and antibodies or other binding molecules(complexes) according to the description, the immunogenicity of theunderlying peptides is secondary. In these cases, the presentation is adetermining factor.

While advances have been made in the development of molecular-targetingdrugs for cancer therapy, there remains a need in the art to develop newanti-cancer agents that specifically target molecules highly specific tocancer cells. The present description addresses that need by providingnovel MAG-003 TCRs, nucleic acids, vectors and host cells whichspecifically bind MAG-003; and methods of using such molecules in thetreatment of cancer.

SUMMARY

The present description relates to T-cell receptors (TCRs) comprising analpha chain and/or a beta chain (“alpha/beta TCRs”). In anotherembodiment, the present description relates to TCRs comprising a gammachain and/or delta chain (“gamma/delta TCRs”).

The present description further relates to TCRs, individual TCR subunits(alone or in combination), and subdomains thereof, soluble TCRs (sTCRs),for example, soluble alpha/beta dimeric TCRs having at least onedisulfide inter-chain bond between constant domain residues that are notpresent in native TCRs, and cloned TCRs, said TCRs engineered intoautologous or allogeneic T-cells or T-cell progenitor cells, and methodsof making same, as well as other cells bearing said TCR.

The present description further relates to a TCR that specifically bindsto a MAG-003 peptide-HLA molecule complex, wherein the MAG-003 peptideis selected from KVLEHVVRV (SEQ ID NO:1) and variants thereof, such asthose shown in SEQ ID NO:2 to SEQ ID NO:24. In an embodiment the HLAmolecule is HLA-A*02.

The present description further relates to TCRs comprising, a TCR alphavariable domain that has at least 75%, 80%, 90%, 95%, 98%, or 99%sequence identity, preferably 90% sequence identity, to a TCR alphavariable domain shown in Table 2; and the TCR beta variable domain hasat least at least 75%, 80%, 90%, 95%, 98%, or 99% sequence identity,preferably 90% sequence identity, to a TCR beta variable domain shown inTable 2.

In an embodiment, the TCR alpha variable domain has at least onemutation relative to a TCR alpha domain shown in Table 2; and/or the TCRbeta variable domain has at least one mutation relative to a TCR alphadomain shown in Table 2. In an embodiment, a TCR comprising at least onemutation in the TCR alpha variable domain and/or TCR beta variabledomain has a binding affinity for, and/or a binding half-life for, aMAG-003 peptide-HLA molecule complex, which is at least double that of aTCR comprising the unmutated TCR alpha domain and/or unmutated TCR betavariable domain.

The TCR alpha chains of the present description may further comprise aTCR alpha constant domain that has at least 70%, 75%, 80%, 90%, 95%,98%, or 99% sequence identity to a TCR alpha constant domain shown inTable 2. The TCR beta chains of the present description may furthercomprise a TCR beta constant domain that has at least 70%, 75%, 80%,90%, 95%, 98%, or 99% sequence identity to a TCR beta constant domainshown in Table 2.

The TCR alpha chains of the present description may further comprise aTCR alpha transmembrane domain and/or a TCR alpha intracellular domain.The TCR beta chains of the present description may further comprise aTCR beta transmembrane domain and/or a TCR beta intracellular domain.

The description further relates to TCR alpha chains comprising one ormore alpha chain complementarity determining regions (CDRs) disclosed inTable 2, and variants thereof having one, two, three or foursubstitutions relative to the CDRs shown in Table 2. Further describedare TCR alpha chains comprising at least one CDR selected from a CDR1,CDR2 and CDR3 shown in Table 2. Further described are TCR alpha chainscomprising an alpha chain CDR3 shown in Table 2.

The description further relates to TCR beta chains comprising one ormore beta chain complementarity determining regions (CDRs) disclosed inTable 2, and variants thereof having one, two, three or foursubstitutions relative to the CDRs shown in Table 2. Further describedare TCR beta chains comprising at least one CDR selected from a betachain CDR1, CDR2 and CDR3 shown in Table 2. Further described are TCRbeta chains comprising a beta chain CDR3 shown in Table 2.

The description further relates to an isolated or recombinant nucleicacid comprising a nucleotide sequence encoding a TCR of the presentdescription. In an embodiment, nucleic acids of the description encode aTCR alpha chain and/or a TCR beta chain as shown in Table 2.

The description further relates to a recombinant expression vectorcomprising a nucleic acid encoding a TCR alpha chain, beta chain, orboth, as described herein.

The description further relates to an isolated host cell comprising arecombinant expression vector expressing a nucleic acid encoding the TCRalpha chain, beta chain, or both, as described herein.

The description further relates to an isolated host cell comprising arecombinant expression vector according to the present description,preferably wherein the cell is a human cell, preferably a peripheralblood lymphocyte (PBL), more preferably a CD4 or CD8 positive Tlymphocyte.

The description further relates to an isolated PBL comprising therecombinant expression vector of the description, wherein the PBL is aCD8+ T-cell or a CD4+ T-cell.

The description further relates to a population of cells comprising atleast one host cell described herein.

The description further relates to TCRs and host cells of the presentdescription for use in the treatment of proliferative diseases, such as,non-small cell lung cancer, small cell lung cancer, renal cell cancer,brain cancer, gastric cancer, colorectal cancer, hepatocellular cancer,pancreatic cancer, prostate cancer, leukemia, breast cancer, Merkel cellcarcinoma, melanoma, ovarian cancer, urinary bladder cancer, uterinecancer, gallbladder and bile duct cancer and esophageal cancer.

In an aspect, the host cell is a CD8+ T-cell or a CD4+ T-celltransfected with a nucleic acid encoding at least one TCR according tothe description, wherein the TCR comprises at least one amino acidsequence disclosed in Table 2. In another aspect such host cells areused in the immunotherapy of small cell lung cancer, non-small cell lungcancer, small cell lung cancer, renal cell cancer, brain cancer, gastriccancer, colorectal cancer, hepatocellular cancer, pancreatic cancer,prostate cancer, leukemia, breast cancer, Merkel cell carcinoma,melanoma, ovarian cancer, urinary bladder cancer, uterine cancer,gallbladder and bile duct cancer and esophageal cancer, and preferablynon-small cell lung cancer.

The description further relates to methods of killing or reducing thenumber of cancer cells comprising contacting the cancer cells with aTCR, nucleic acid, vector or host cell as described herein. Alsoprovided are methods of treating cancer comprising administering to asubject in need thereof a TCR, nucleic acid, vector or host cell asdescribed herein.

In another aspect, the present description relates to a MAG-003 peptide,for example an isolated peptide, comprising an amino acid sequenceaccording to the following general formula I:

(SEQ ID NO: 25) X₁X₂LEHVVRX₃ Formula Iwherein X₁ is selected from the amino acids K and Y, X₂ is selected fromthe amino acids V, L and A, and X₃ is selected from V, L, A, and I,wherein said peptide binds to an HLA class I or class II molecule and/orinduces T-cells cross-reacting with said peptide, or a pharmaceuticallyacceptable salt thereof. In an aspect, said peptide is not theunderlying full-length polypeptide.

Preferred is a sequence selected from the group consisting of SEQ IDNO:1 or a variant sequence thereof which is at least 66%, preferably atleast 77%, and more preferably at least 88% homologous (preferably atleast 77% or at least 88% identical) to SEQ ID NO:1, wherein saidvariant binds to an HLA class I or class II molecule and/or inducesT-cells cross-reacting with said peptide, or a pharmaceuticallyacceptable salt thereof, wherein said peptide is not the underlyingfull-length polypeptide.

The present description further relates to a peptide of the presentdescription comprising a sequence that is selected from the groupconsisting of SEQ ID NO:1 or a variant thereof, which is at least 66%,preferably at least 77%, and more preferably at least 88% homologous(preferably at least 77% or at least 88% identical) to SEQ ID NO:1,wherein said peptide or variant thereof has an overall length of between8 and 100, preferably between 8 and 30, and most preferably of between 8and 14 amino acids.

The following tables show example peptides according to the presentdescription, and their respective SEQ ID NOs. In an aspect, the peptidesin Table 1 may bind to HLA-A*02. In another aspect, TCRs describedherein are capable of binding or specifically binding to one or morepeptides in Table 1.

TABLE 1 Peptides according to the present description SEQ ID NO:Sequence 1 KVLEHVVRV 2 KVLEHVVRL 3 KVLEHVVRA 4 KVLEHVVRI 5 KLLEHVVRV 6KLLEHVVRL 7 KLLEHVVRA 8 KLLEHVVRI 9 KALEHVVRV 10 KALEHVVRL 11 KALEHVVRA12 KALEHVVRI 13 YLLEHVVRV 14 YLLEHVVRL 15 YLLEHVVRA 16 YLLEHVVRI 17YALEHVVRV 18 YALEHVVRL 19 YALEHVVRA 20 YALEHVVRI 21 YVLEHVVRV 22YVLEHVVRL 23 YVLEHVVRA 24 YVLEHVVRI

In an aspect, the present description relates to the treatment ofproliferative diseases, such as non-small cell lung cancer, small celllung cancer, renal cell cancer, brain cancer, gastric cancer, colorectalcancer, hepatocellular cancer, pancreatic cancer, prostate cancer,leukemia, breast cancer, Merkel cell carcinoma, melanoma, ovariancancer, urinary bladder cancer, uterine cancer, gallbladder and bileduct cancer and esophageal cancer by utilizing one or more peptidesdescribed herein.

Particularly preferred are the peptides—alone or incombination—according to the present description selected from the groupconsisting of SEQ ID NO:1 to SEQ ID NO:24. More preferred are thepeptides—alone or in combination—selected from the group consisting ofSEQ ID NO:1 to SEQ ID NO:24 (see Table 1), and their uses in theimmunotherapy of non-small cell lung cancer, small cell lung cancer,renal cell cancer, brain cancer, gastric cancer, colorectal cancer,hepatocellular cancer, head and neck cancer, pancreatic cancer, prostatecancer, leukemia, breast cancer, Merkel cell carcinoma, melanoma,ovarian cancer, urinary bladder cancer, uterine cancer, gallbladder andbile duct cancer and esophageal cancer, and preferably non-small celllung cancer.

In an aspect, the present description relates to peptides according tothe present description that have the ability to bind to a molecule ofHLA class-I or —in a longer form, such as a length-variant—HLA class-II.

In an aspect, the present description relates to the peptides accordingto the present description wherein said peptides (each) comprise,consist of, or consist essentially of an amino acid sequence accordingto SEQ ID NO:1 to SEQ ID NO:24.

The present description further relates to the peptides according to thepresent description, wherein said peptide is modified and/or includesnon-peptide bonds.

The present description further relates to the peptides according to thepresent description, wherein said peptide is part of a fusion protein,in particular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), preferably the 80 N-terminalamino acids thereof, or fused to (or into the sequence of) an antibody,such as, for example, an antibody that is specific for dendritic cells.

The description further relates to a nucleic acid encoding the peptidesaccording to the description. The description further relates to thenucleic acid according to the description that is DNA, cDNA, PNA, RNA orcombinations thereof.

The description further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to thedescription.

The description further relates to a peptide according to thedescription, a nucleic acid according to the description or anexpression vector according to the description for use in the treatmentof diseases and in medicine, in particular in the treatment of cancer.

The description further relates to antibodies that specifically bind thepeptides according to the present description or complexes of saidpeptides according to the description with MHC, and methods of makingsuch antibodies.

The description further relates to a host cell comprising a nucleic acidaccording to the description or an expression vector as describedbefore.

The description further relates to the host cell according to thedescription that is an antigen presenting cell, and preferably is adendritic cell.

The description further relates to a method for producing a peptideaccording to the description, the method comprising culturing the hostcell according to the description, and isolating the peptide from saidhost cell or its culture medium.

The description further relates to methods according to the description,wherein the antigen is loaded onto class I or II MHC molecules expressedon the surface of a suitable antigen-presenting cell or artificialantigen-presenting cell by contacting a sufficient amount of the antigenwith an antigen-presenting cell or the antigen is loaded onto class I orII MHC tetramers by tetramerizing the antigen/class I or II MHC complexmonomers.

The description further relates to the method according to thedescription, wherein the antigen-presenting cell comprises an expressionvector capable of expressing or expressing said peptide containing SEQID NO:1 to SEQ ID NO:24, preferably containing SEQ ID NO:1 to SEQ IDNO:24, or a variant amino acid sequence thereof.

The description further relates to activated T-lymphocytes, produced bythe method according to the description, wherein a T-cell selectivelyrecognizes a cell which expresses a polypeptide comprising an amino acidsequence according to the description.

The description further relates to methods of killing cancer and/orsuppressing cells in a patient which cancer cells aberrantly express apolypeptide comprising any amino acid sequence according to thedescription, the methods comprising administering to the patient aneffective number of T-cells as produced according to the description.

The description further relates to the use of any peptide describedherein, nucleic acids according described herein, expression vectorsdescribed herein, cells described herein, activated T lymphocytedescribed herein, T-cell receptors, antibodies, or other peptide- and/orpeptide-MHC-binding molecules according to the present description as amedicament or in the manufacture of a medicament. In an aspect, themedicament is active against cancer.

Preferably, said medicament is a cellular therapy, a vaccine or aprotein based on a TCR, a soluble TCR or antibody.

The present description further relates to a use according to thepresent description, wherein the cancer cells are non-small cell lungcancer, small cell lung cancer, renal cell cancer, brain cancer, gastriccancer, colorectal cancer, hepatocellular cancer, pancreatic cancer,prostate cancer, leukemia, breast cancer, Merkel cell carcinoma,melanoma, ovarian cancer, urinary bladder cancer, uterine cancer,gallbladder and bile duct cancer and esophageal cancer, and preferablynon-small cell lung cancer.

The present description further relates to biomarkers based on thepeptides according to the present description, herein called “targets,”that can be used in the diagnosis of cancer, preferably non-small celllung cancer. The marker can be the over-presentation of the peptide(s)themselves, or over-expression of the corresponding gene(s). The markersmay also be used to predict the probability of success of a treatment,preferably an immunotherapy, and most preferred an immunotherapytargeting the same target that is identified by the biomarker. Forexample, an antibody or soluble TCR can be used to stain sections of thetumor to detect the presence of a peptide of interest in complex withMHC. Optionally the antibody or soluble TCR carries a further effectorfunction such as an immune stimulating domain or toxin.

The present description further relates to the use of these noveltargets for the identification of TCRs that recognize at least one ofsaid targets, and preferably the identification of said TCRs thatactivate T-cells.

The present description also relates to the use of these novel targetsin the context of cancer treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows MAG-003 peptide presentation in healthy tissues andcancers.

FIG. 2 shows MAG-003 expression in cancer and healthy tissues.

FIG. 3 shows MAG-003 expression in cancer and healthy tissues.

FIG. 4 shows MAG-003 expression in cancer and healthy tissues.

FIG. 5 shows IFNγ release from CD8+ T-cells electroporated with alphaand beta chain RNA of TCRs R7P1D5, R20P1H7 and R10P2G12 (Table 2),respectively, after co-incubation with target cells loaded with MAG-003peptide (SEQ ID NO:1) or homologous but unrelated peptide RABGAP1L-001(SEQ ID NO:91), AXIN1-001 (SEQ ID NO:92), ANO5-001 (SEQ ID NO:93),TPX2-001 (SEQ ID NO:94), SYNE3-001 (SEQ ID NO:95), MIA3-001 (SEQ IDNO:96), HERC4-001 (SEQ ID NO:97), PSME2-001 (SEQ ID NO:98), HEATR5A-001(SEQ ID NO:99) or CNOT1-003 (SEQ ID NO:100) or control peptideNYESO1-001 (SEQ ID NO:101). IFNγ release data were obtained with CD8+T-cells derived from two different donors (donor 1 on right X-axis,donor 2 on left X-axis). RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls.

FIG. 6 shows IFNγ release from CD8+ T-cells electroporated with alphaand beta chain RNA of TCRs R7P1 D5, R20P1H7 and R10P2G12 (Table 2),respectively, after co-incubation with target cells loaded with MAG-003peptide (SEQ ID NO:1) or homologous but unrelated peptide RABGAP1L-001(SEQ ID NO:91), AXIN1-001 (SEQ ID NO:92), ANO5-001 (SEQ ID NO:93),TPX2-001 (SEQ ID NO:94), SYNE3-001 (SEQ ID NO:95), MIA3-001 (SEQ IDNO:96), HERC4-001 (SEQ ID NO:97), PSME2-001 (SEQ ID NO:98), HEATR5A-001(SEQ ID NO:99) or CNOT1-003 (SEQ ID NO:100) or control peptideNYESO1-001 (SEQ ID NO:101). IFNγ release data were obtained with CD8+T-cells derived from two different donors (donor 1 on right X-axis,donor 2 on left X-axis). RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls.

FIG. 7 shows IFNγ release from CD8+ T-cells electroporated with alphaand beta chain RNA of TCRs R7P1 D5, R20P1H7 and R10P2G12 (Table 2),respectively, after co-incubation with target cells loaded with MAG-003peptide (SEQ ID NO:1) or homologous but unrelated peptide RABGAP1L-001(SEQ ID NO:91), AXIN1-001 (SEQ ID NO:92), ANO5-001 (SEQ ID NO:93),TPX2-001 (SEQ ID NO:94), SYNE3-001 (SEQ ID NO:95), MIA3-001 (SEQ IDNO:96), HERC4-001 (SEQ ID NO:97), PSME2-001 (SEQ ID NO:98), HEATR5A-001(SEQ ID NO:99) or CNOT1-003 (SEQ ID NO:100) or control peptideNYESO1-001 (SEQ ID NO:101). IFNγ release data were obtained with CD8+T-cells derived from two different donors (donor 1 on right X-axis,donor 2 on left X-axis). RNA electroporated CD8+ T-cells alone or inco-incubation with unloaded target cells served as controls.

FIG. 8 show MHC/MAG-003 tetramer or MHC/NYESO1-001 tetramer staining ofCD8+ T-cells electroporated with alpha and beta chain RNA of TCRs R7P1D5, R20P1H7 and R10P2G12 (Table 2), respectively. CD8+ T-cellselectroporated with RNA of 1G4 TCR that specifically binds toMHC/NYESO1-001 complex and mock electroporated CD8+ T-cells served ascontrols.

FIG. 9 shows IFNγ release from CD8+ T-cells electroporated with alphaand beta chain RNA of TCRs R7P1 D5, R20P1H7 and R10P2G12 (Table 2),respectively, after co-incubation with target cells loaded with MAG-003peptide (SEQ ID NO:1) or various MAG-003 alanine-substitution variantsat positions 1-9 of SEQ ID NO:1. RNA-electroporated CD8+ T-cells aloneor in co-incubation with target cells loaded with control peptideNYESO1-001 or unloaded target cells served as controls. IFNγ releasedata were obtained with CD8+ T-cells derived from two different donors(donor 1 on right X-axis, donor 2 on left X-axis).

FIG. 10 shows IFNγ release from CD8+ T-cells electroporated with alphaand beta chain RNA of TCRs R7P1 D5, R20P1H7 and R10P2G12 (Table 2),respectively, after co-incubation with target cells loaded with MAG-003peptide (SEQ ID NO:1) or various MAG-003 alanine-substitution variantsat positions 1-9 of SEQ ID NO:1. RNA-electroporated CD8+ T-cells aloneor in co-incubation with target cells loaded with control peptideNYESO1-001 or unloaded target cells served as controls. IFNγ releasedata were obtained with CD8+ T-cells derived from two different donors(donor 1 on right X-axis, donor 2 on left X-axis).

FIG. 11 shows IFNγ release from CD8+ T-cells electroporated with alphaand beta chain RNA of TCRs R7P1 D5, R20P1H7 and R10P2G12 (Table 2),respectively, after co-incubation with target cells loaded with MAG-003peptide (SEQ ID NO:1) or various MAG-003 alanine-substitution variantsat positions 1-9 of SEQ ID NO:1. RNA-electroporated CD8+ T-cells aloneor in co-incubation with target cells loaded with control peptideNYESO1-001 or unloaded target cells served as controls. IFNγ releasedata were obtained with CD8+ T-cells derived from two different donors(donor 1 on right X-axis, donor 2 on left X-axis).

FIG. 12 IFNγ release from CD8+ T-cells electroporated with alpha andbeta chain RNA of TCRs (A) R7P1 D5, (B) R20P1H7, and (C) R10P2G12 afterco-incubation with A-375 melanoma cell line, T98G glioblastoma cell lineand SK-BR-3 breast cancer cell line, respectively. RNA-electroporatedCD8+ T-cells alone served as a control.

FIG. 13 T cells transfected with RNA coding for TCR R7P1 D5, R10P2G12,R20P1H7, mock or the control TCR 1G4 (SEQ ID NO:X) were co-cultured withdifferent primary human healthy tissue cells (see table 13 forabbreviations), the target negative tumor cell line MCF-7 and theMAGEA4- and NY-ESO1-positive cell lines A-375 and NCI-H1755. TCRsR7P1D5, R10P2G12, R20P1H7 and 1G4 expression in T cells was achieved byelectroporation using an RNA vector. Human healthy tissue cell culturesdid not lead to significant TCR-mediated recognition and T-cellactivation for TCR R7P1D5, R10P2G12 or R20P1H7. E:T ratio 1:1, 20,000cells each, mean of IFN-γ release after 20 h from replicates is shown asmeasured by ELISA. Error bars indicate standard deviations. Results werenormalized by subtraction of IFN-γ release from TCR-transfected T cellsonly and IFN-γ release due to co-culture of Target cells and T cells notexpressing TCR of interest.

FIG. 14 shows cumulative data from 4 donors screened for R7P1 D5 TCRtransgene expression after transduction with 6 different lentiviralconstructs (R71-R78). CD3+/CD8+ T-cells were analyzed by flow cytometryfor proportion of MHC/MAG-003 tetramer binding cells at 96 hours posttransduction. Non-transduced T-cells (NT) stained MHC/MAG-003 tetramerand 1G4 (NY-ESO1) TCR transduced T-cells stained with MHC/NYESO1-001tetramer were used as negative and positive control, respectively. Cellswere gated on Live CD3+/CD8+ population.

FIG. 15A shows effector response of R7P1 D5 TCR transduced T-cellsmeasured by means of IFNγ production upon co-culture MAGEA4 expressingtumor cell line A375 and MAGEA4 negative tumor cell line MCF-7. T-cellstransduced with concentrated supernatant of R73 lentivirus encoding R7P1D5 TCR were compared with non-transduced cells (NT) both derived fromPBMCs of 4 healthy donors. Results are presented as mean±SEM oftriplicates.

FIG. 15B shows IFNγ response of R7P1D5 TCR transduced (R73 lentivirus)T-cells from PBMC of 2 healthy donors (Donor 6 and 7) upon co-incubationwith T2 target cells pulsed with decreasing concentrations of MAG-003peptide. Results are presented as mean±SEM of triplicates measured 96hours after transduction.

FIG. 16 shows cytotoxic response of R7P1 D5 TCR (R73 lentivirus)transduced and non-transduced T-cells (NT) measured against MAGEA4expressing tumor cell line A375 at different E:T ratios in a 4h Cr51release assay. Results are presented as mean±SEM of 3-4 replicates. (C)Combined data from 7 R7P1 D5 TCR (R73 lentivirus) transduced T-cellproducts generated from 5 donors showing antigen specific killing ofMAGEA4 expressing (A375) and MAGEA4-negative (MCF-7) tumor targets atE:T ration of 40:1.

DETAILED DESCRIPTION

The present description relates to T-cell receptors (TCRs) comprising analpha chain and/or a beta chain (“alpha/beta TCRs”). Also provided areMAG-003 peptides capable of binding to TCRs and antibodies whenpresented by an MHC molecule. The present description also relates tonucleic acids, vectors and host cells for expressing TCRs and peptidesof the present description; and methods of using the same.

The object of the invention is therefore solved in a first aspect by anantigen recognizing construct comprising at least one complementarydetermining region (CDR) 3 having at least 50%, 60%, 70%, 80%, 90%, 95%,98%, 99%, or preferably 100% sequence identity to an amino acid sequenceselected from SEQ ID NOs. 44, 52, 60, 68, 76, and 84.

The antigen recognizing construct according to the invention ispreferably selected from an antibody, or derivative or fragment thereof,or a T cell receptor (TCR), or derivative or fragment thereof. Aderivative or fragment of an antibody or TCR of the invention shallpreferably retain the antigen binding/recognizing ability of the parentmolecule, in particular its specificity and/or selectivity as explainedabove. Such binding functionality may be retained by the presence of aCDR3 region as defined herein.

In an embodiment of the invention, the inventive TCRs are able torecognize TAA antigens in a major histocompatibility complex (MHC) classI-dependent manner. “MHC class I-dependent manner,” as used herein,means that the TCR elicits an immune response upon binding to TAAantigens within the context of an MHC class I molecule. The MHC class Imolecule can be any MHC class I molecule known in the art, e.g., HLA-Amolecules. In a preferred embodiment of the invention, the MHC class Imolecule is an HLA-A*02 molecule.

The invention provides both single chain antigen recognizing constructand double chain recognizing constructs.

In an embodiment, the TCR alpha variable domain has at least onemutation relative to a TCR alpha domain shown in Table 2; and/or the TCRbeta variable domain has at least one mutation relative to a TCR alphadomain shown in Table 2. In an embodiment, a TCR comprising at least onemutation in the TCR alpha variable domain and/or TCR beta variabledomain has a binding affinity for, and/or a binding half-life for, anTAA peptide-HLA molecule complex, which is at least double that of a TCRcomprising the unmutated TCR alpha domain and/or unmutated TCR betavariable domain.

The TCR alpha chains of the present description may further comprise aTCR alpha transmembrane domain and/or a TCR alpha intracellular domain.The TCR beta chains of the pre-sent description may further comprise aTCR beta transmembrane domain and/or a TCR beta intracellular domain.

The invention in particular provides a TCR as antigen recognizingconstruct, or fragment or derivative thereof. The TCR preferably is ofhuman, which is understood as being generated from a human TCR locus andtherefore comprising human TCR sequences. Furthermore, the TCR of theinvention may be characterized in that it is of human origin andspecifically recognizes a TAA antigen of the invention as mentioned intable 1.

Another embodiment of the invention additionally or alternativelyprovides the antigen recognizing construct described above, whichinduces an immune response, preferably wherein the immune response ischaracterized by an increase in interferon (IFN) γ levels.

TCRs of the invention may be provided as single chain α or β, or γ andδ, molecules, or alternatively as double chain constructs composed ofboth the α and β chain, or γ and δ chain.

Most preferably, in some additional embodiments, wherein the disclosurerefers to antigen recognizing constructs comprising any one, two or allof the CDR1 to CDR3 regions of the herein disclosed TCR chains (seeTable 1), such antigen recognizing constructs may be preferred, whichcomprise the respective CDR sequence of the invention with not more thanthree, two, and preferably only one, modified amino acid residues. Amodified amino acid residue may be selected from an amino acidinsertion, deletion or substitution. Most preferred is that the three,two, preferably only one modified amino acid residue is the first orlast amino acid residue of the respective CDR sequence. If themodification is a substitution then it is preferable in some embodimentsthat the substitution is a conservative amino acid substitution.

The inventive TCRs may further comprise a constant region derived fromany suitable species, such as any mammal, e.g., human, rat, monkey,rabbit, donkey, or mouse. In an embodiment of the invention, theinventive TCRs further comprise a human constant region. In somepreferred embodiments, the constant region of the TCR of the inventionmay be slightly modified, for example, by the introduction ofheterologous sequences, preferably mouse sequences, which may increaseTCR expression and stability.

In an embodiment of the invention, chimeric TCR are provided, whereinthe TCR chains comprise sequences from multiple species. Preferably, aTCR of the invention may comprise an α chain comprising a human variableregion of an α chain and, for example, a murine constant region of amurine TCR α chain.

In one embodiment, the TCR of the invention is a human TCR comprisinghuman variable regions according to the above embodiments and humanconstant regions.

In some embodiments the antigen recognizing construct is murinized orhumanized. These terms are used when amino acid sequences from a foreignspecies are introduced into a con-struct of the invention.

The TCR of the invention may be provided as a single chain TCR (scTCR).A scTCR according to the invention shall comprise in one polypeptidechain α full or partial alpha chain sequence and a full or partial betachain sequence, preferably connected via a peptide linker.

A scTCR can comprise a polypeptide of a variable region of a first TCRchain (e.g., an alpha chain) and a polypeptide of an entire(full-length) second TCR chain (e.g., a beta chain), or vice versa.Furthermore, the scTCR can optionally comprise one or more linkers whichjoin the two or more polypeptides together. The linker can be, forinstance, a peptide, which joins together two single chains, asdescribed herein. Also provided is such a scTCR of the invention, whichis fused to a human cytokine, such as IL-2, IL-7 or IL-15.

The antigen recognizing construct according to the invention can also beprovided in the form of a multimeric complex, comprising at least twoscTCR molecules, wherein said scTCR molecules are each fused to at leastone biotin moiety, or other interconnecting molecule/linker, and whereinsaid scTCRs are interconnected by biotin-streptavidin interaction toallow the formation of said multimeric complex. Similar approaches knownin the art for the generation of multimeric TCR are also possible andincluded in this disclosure. Also provided are multimeric complexes of ahigher order, comprising more than two scTCR of the invention.

For the purposes of the present invention, a TCR is a moiety having atleast one TCR alpha or gamma and/or TCR beta or delta variable domain.Generally, they comprise both a TCR alpha variable domain and a TCR betavariable domain, alternatively both a TCR gamma variable domain and aTCR delta variable domain. They may be αβ/γδ heterodimers or may be insingle chain format. For use in adoptive therapy, an αβ or γδheterodimeric TCR may, for example, be transfected as full length chainshaving both cytoplasmic and transmembrane domains. If desired, anintroduced disulfide bond between residues of the respective constantdomains may be present.

In a preferred embodiment, the antigen recognizing construct is a humanTCR, or fragment or derivative thereof. A human TCR or fragment orderivative thereof is a TCR, which comprises over 50% of thecorresponding human TCR sequence. Preferably, only a small part of theTCR sequence is of artificial origin or derived from other species. Itis known, however, that chimeric TCRs, e.g. derived from human originwith murine sequences in the constant do-mains, are advantageous.Particularly preferred are, therefore, TCRs in accordance with thepresent invention, which contains murine sequences in the extracellularpart of their constant domains.

Thus, it is also preferred that the inventive antigen recognizingconstruct is able to recognize its antigen in a human leucocyte antigen(HLA) dependent manner, preferably in a HLA-A02 dependent manner. Theterm “HLA dependent manner” in the context of the present inventionmeans that the antigen recognizing construct binds to the antigen onlyin the event that the antigenic peptide is presented by said HLA.

The antigen recognizing construct in accordance with the invention inone embodiment preferably induces an immune response, preferably whereinthe immune response is characterized by the increase in interferon (IFN)γ levels.

Also provided by the invention is a polypeptide comprising a functionalportion of any of the TCRs (or functional variants thereof) describedherein, for examples, of any one of the TCRs as provided in the examplesection and table 2. The term “poly-peptide” as used herein includesoligopeptides and refers to a single chain of amino acids connected byone or more peptide bonds. With respect to the inventive polypeptides,the functional portion can be any portion comprising contiguous aminoacids of the TCR (or functional variant thereof), of which it is a part,provided that the functional portion specifically binds to the TAAantigen, preferably as disclosed herein in Table 1. The term “functionalportion” when used in reference to a TCR (or functional variant thereof)refers to any part or fragment of the TCR (or functional variantthereof) of the invention, which part or fragment retains the biologicalactivity of the TCR (or functional variant thereof), of which it is apart (the parent TCR or parent functional variant thereof). Functionalportions encompass, for example, those parts of a TCR (or functionalvariant thereof) that retain the ability to specifically bind to the TAAantigen (in an HLA dependent manner), or detect, treat, or preventcancer, to a similar extent, the same extent, or to a higher extent, asthe parent TCR (or functional variant thereof). In reference to theparent TCR (or functional variant thereof), the functional portion cancomprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, ormore, of the parent TCR variable sequences (or functional variantthereof).

The functional portion can comprise additional amino acids at the aminoor carboxy terminus of the portion, or at both termini, in whichadditional amino acids are not found in the amino acid sequence of theparent TCR or functional variant thereof. Desirably, the additionalamino acids do not interfere with the biological function of thefunctional portion, e.g., specifically binding to the TAA antigens;and/or having the ability to detect cancer, treat or prevent cancer,etc. More desirably, the additional amino acids enhance the biologicalactivity, as compared to the biological activity of the parent TCR orfunctional variant thereof.

In some instances, the construct of the invention may comprise one ortwo polypeptide chains comprising a sequences according to any of theSEQ ID NO: 39 to 86 (CDR sequences, constant and variable regions andfull length sequences), or functional fragments thereof, and furthercomprise(s) other amino acid sequences, e.g., an amino acid sequenceencoding an immunoglobulin or a portion thereof, then the inventiveprotein can be a fusion protein. In this regard, the invention alsoprovides a fusion protein comprising at least one of the inventivepolypeptides described herein along with at least one other polypeptide.The other polypeptide can exist as a separate polypeptide of the fusionprotein, or can exist as a polypeptide, which is expressed in frame (intandem) with one of the inventive polypeptides described herein. Theother polypeptide may include any peptidic or proteinaceous molecule, ora portion thereof, including, but not limited to an immunoglobulin, CD3,CD4, CD8, an MHC molecule, a CD1 molecule, e.g., CD1a, CD1b, CD1c, CD1d,etc.

The fusion protein can comprise one or more copies of the inventivepolypeptide and/or one or more copies of the other polypeptide. Forinstance, the fusion protein can comprise 1, 2, 3, 4, 5, or more, copiesof the inventive polypeptide and/or of the other polypeptide. Suitablemethods of making fusion proteins are known in the art, and include, forexample, recombinant methods. In some embodiments of the invention, theTCRs (and functional portions and functional variants thereof),polypeptides, and proteins of the invention may be expressed as a singleprotein comprising a linker peptide linking the α chain and the β chain,and linking the γ chain and the δ chain. In this regard, the TCRs (andfunctional variants and functional portions thereof), polypeptides, andproteins of the invention comprising the amino acid sequences of thevariable regions of the TCR of the invention and may further comprise alinker pep-tide. The linker peptide may advantageously facilitate theexpression of a recombinant TCR (including functional portions andfunctional variants thereof), polypeptide, and/or protein in a hostcell. The linker peptide may comprise any suitable amino acid sequence.Linker sequences for single chain TCR constructs are well known in theart. Such a single chain construct may further comprise one, or two,constant domain sequences. Upon expression of the construct includingthe linker peptide by a host cell, the linker peptide may also becleaved, resulting in separated α and β chains, and separated γ and δchain.

As already mentioned above, the binding functionality of the TCR of theinvention may be provided in the framework of an antibody. For example,CDR sequences of the TCR of the invention, possibly including additional3, 2 or 1 N and/or C terminal framework residues, may be directlygrafted into an antibody variable heavy/light chain sequence. The term“antibody” in its various grammatical forms is used herein to refer toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain anantigen-binding site or a paratope. Such molecules are also referred toas “antigen binding fragments” of immunoglobulin molecules. Theinvention further provides an antibody, or antigen binding portionthereof, which specifically binds to the antigens described herein. Theantibody can be any type of immunoglobulin that is known in the art. Forinstance, the antibody can be of any isotype, e.g., IgA, IgD, IgE, IgG,IgM, etc. The antibody can be monoclonal or polyclonal. The antibody canbe a naturally-occurring antibody, e.g., an antibody isolated and/orpurified from a mammal, e.g., mouse, rabbit, goat, horse, chicken,hamster, human, etc. Alternatively, the antibody can be agenetically-engineered antibody, e.g., a humanized antibody or achimeric antibody. The antibody can be in monomeric or pol-ymeric form.

The term “antibody” includes, but is not limited to, geneticallyengineered or otherwise modified forms of immunoglobulins, such asintrabodies, chimeric antibodies, fully human antibodies, humanizedantibodies (e.g. generated by “CDR-grafting”), antibody fragments, andheteroconjugate antibodies (e.g., bispecific antibodies, diabodies,triabodies, tetra-bodies, etc.). The term “antibody” includescys-diabodies and minibodies. Thus, each and every embodiment providedherein in regard to “antibodies”, or “antibody like constructs” is alsoenvisioned as, bi-specific antibodies, diabodies, scFv fragments,chimeric antibody receptor (CAR) constructs, diabody and/or minibodyembodiments, unless explicitly denoted otherwise. The term “antibody”includes a polypeptide of the immunoglobulin family or a polypeptidecomprising fragments of an immunoglobulin that is capable ofnon-covalently, reversibly, and in a specific manner binding acorresponding antigen, preferably the TAA of the invention, as disclosedherein. An exemplary antibody structural unit comprises a tetramer. Insome embodiments, a full length antibody can be composed of twoidentical pairs of polypeptide chains, each pair having one “light” andone “heavy” chain (connected through a disulfide bond). Antibodystructure and isotypes are well known to the skilled artisan (forexample from Janeway's Immunobiology, 9th edition, 2016).

The recognized immunoglobulin genes of mammals include the kappa,lambda, alpha, gamma, delta, epsilon, and mu constant region genes, aswell as the myriad immunoglobulin variable region genes (for moreinformation on immunoglobulin genes see the internationalIm-MunoGeneTics information System®, Lefranc M-P et al, Nucleic AcidsRes. 2015 January; 43(Database issue):D413-22; and www.imgt.org). Forfull-length chains, the light chains are classified as either kappa orlambda. For full-length chains, the heavy chains are classified asgamma, mu, alpha, delta, or epsilon, which in turn define theimmunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. TheN-terminus of each chain defines a variable region of about 100 to 110or more amino acids primarily responsible for antigen recognition. Theterms variable light chain (VL) and variable heavy chain (VH) refer tothese regions of light and heavy chains respectively. As used in thisinvention, an “antibody” en-compasses all variations of antibody andfragments thereof. Thus, within the scope of this concept are fulllength antibodies, chimeric antibodies, humanized antibodies, singlechain antibodies (scFv), Fab, Fab′, and multimeric versions of thesefragments (e.g., F(ab′)2) with the same, essentially the same or similarbinding specificity. In some embodiments, the anti-body bindsspecifically to a peptide TAA of the invention. Preferred antigenrecognizing constructs according to the invention include an antibodyheavy chain, preferably the variable domain thereof, or an antigenbinding fragment thereof, and/or an antibody light chain, preferably thevariable domain thereof, or an antigen binding fragment thereof.Similarly, disulfide-stabilized variable region fragments (dsFv) can beprepared by recombinant DNA technology, antibody fragments of theinvention, however, are not limited to these exemplary types of antibodyfragments. Also, the antibody, or antigen binding portion thereof, canbe modified to comprise a detectable label, such as, for instance, aradioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC),phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradishperoxidase), and element particles (e.g., gold particles). In someinstances, the TCR CDR3 sequence may be slightly modified, butpreferably by not more than 3 amino acid residues, preferably only twoand most preferably only one amino acid position, as compared to theCDR3 sequences provided in SEQ ID Nos: 44, 52, 60, 68, 76, and 84.Preferably, the antibodies comprise the CDR3, preferably all of CDR1 toCDR3 regions in the combination, as indicated for the TCR of theinvention in table 2, in each case independently, optionally with notmore than three or two, preferably one, amino acid substitution(s),insertion(s) and/or deletion(s) compared to these sequences.

Suitable methods of making antibodies are known in the art. Forinstance, standard hybridoma methods are described in, e.g., Kohler andMilstein, Eur. J. Immunol, 5, 511-519 (1976), Harlow and Lane (eds.),Antibodies: A Laboratory Manual, CSH Press (1988), and C. A. Janeway etal. (eds.), Immunobiology, 8 Ed., Garland Publishing, New York, N.Y.(2011)). Alternatively, other methods, such as EBV-hybridoma methods(Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984), andRoder et al, Methods Enzymol, 121, 140-67 (1986)), and bacteriophagevector expression systems (see, e.g., Huse et al., Science, 246, 1275-81(1989)) are known in the art. Further, methods of producing antibodiesin non-human animals are described in, e.g., U.S. Pat. Nos. 5,545,806,5,569,825, and 5,714,352, and U.S. Patent Application Publication No.2002/0197266.

Some embodiments of the invention also pertain to TCRs, or functionalfragments and polypeptides thereof, which are soluble TCRs. As usedherein, the term “soluble T-cell receptor” refers to heterodimerictruncated variants of native TCRs, which comprise extracellular portionsof the TCR α-chain and β-chain, for example linked by a disulfide bond,but which lack the transmembrane and cytosolic domains of the nativeprotein. The terms “soluble T-cell receptor α-chain sequence and solubleT-cell receptor β-chain sequence” refer to TCR α-chain and β-chainsequences that lack the transmembrane and cytosolic domains. Thesequence (amino acid or nucleic acid) of the soluble TCR α-chain andβ-chains may be identical to the corresponding sequences in a native TCRor may comprise variant soluble TCR α-chain and β-chain sequences, ascompared to the corresponding native TCR sequences. The term “solubleT-cell receptor” as used herein encompasses soluble TCRs with variant ornon-variant soluble TCR α-chain and β-chain sequences. The variationsmay be in the variable or constant regions of the soluble TCR α-chainand β-chain sequences and can include, but are not limited to, aminoacid deletion, insertion, substitution mutations as well as changes tothe nucleic acid sequence, which do not alter the amino acid sequence.Soluble TCR of the invention in any case retain the bindingfunctionality of their parent molecules.

Definitions

As used herein and except as noted otherwise all terms are defined asgiven below.

The term “T-cell receptor” (abbreviated TCR) refers to a heterodimericmolecule comprising an alpha polypeptide chain (alpha chain) and a betapolypeptide chain (beta chain), wherein the heterodimeric receptor iscapable of binding to a peptide antigen presented by an HLA molecule.

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T-cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target-cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, or 12 or longer, and incase of MHC class II peptides (longer variants of the peptides of thedescription) they can be as long as 13, 14, 15, 16, 17, 18, 19 or 20 ormore amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present description differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the description, as longas the correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the description as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentdescription), if it is capable of inducing an immune response. In thecase of the present description, immunogenicity is more specificallydefined as the ability to induce a T-cell response. Thus, an “immunogen”would be a molecule that is capable of inducing an immune response, andin the case of the present description, a molecule capable of inducing aT-cell response. In another aspect, the immunogen can be the peptide,the complex of the peptide with MHC, oligopeptide, and/or protein thatis used to raise specific antibodies or TCRs against it.

A class I T-cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by aT-cell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this description are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein the term “a nucleotide coding for (or encoding) a TCR”refers to one or more nucleotide sequences coding for the TCR includingartificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example,T-cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. In an aspect,such polynucleotides are part of a vector and/or such polynucleotides orpolypeptides are part of a composition, and still are isolated in thatsuch vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present description may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosedaccording to the present description, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present description can advantageously be in enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present description, the term “percent identity”or “percent identical”, when referring to a sequence, means that asequence is compared to a claimed or described sequence after alignmentof the sequence to be compared (the “Compared Sequence”) with thedescribed or claimed sequence (the “Reference Sequence”). The percentidentity is then determined according to the following formula:

percent identity=100 [1−(C/R)]

wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and(ii) each gap in the Reference Sequence and(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and(iv) the alignment has to start at position 1 of the aligned sequences;and R is the number of bases or amino acids in the Reference Sequenceover the length of the alignment with the Compared Sequence with any gapcreated in the Reference Sequence also being counted as a base or aminoacid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

In the description, the term “homologous” refers to the degree ofidentity (see percent identity above) between sequences of two aminoacid sequences, i.e., peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T-cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide having the given amino acidsequence selected from the group consisting of SEQ ID NO:1 to SEQ IDNO:24. For example, a peptide may be modified so that it at leastmaintains, if not improves, the ability to interact with and bind to thebinding groove of a suitable MHC molecule, such as HLA-A*02 or -DR, andin that way it at least maintains, if not improves, the ability to bindto the TCR of activated T-lymphocytes. Similarly, a TCR may be modifiedso that it at least maintains, if not improves, the ability to interactwith and bind to a suitable MHC molecule/KVLEHVVRV (SEQ ID NO:1)complex, such as HLA-A*02 or -DR, and in that way it at least maintains,if not improves, the ability to activate T-cells.

These T-cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide, such as KVLEHVVRV (SEQ ID NO:1), as defined inthe aspects of the description. As can be derived from the scientificliterature and databases (Rammensee et al., 1999; Godkin et al., 1997),certain positions of HLA binding peptides are typically anchor residuesforming a core sequence fitting to the binding motif of the HLAreceptor, which is defined by polar, electrophysical, hydrophobic andspatial properties of the polypeptide chains constituting the bindinggroove. In an aspect, one skilled in the art would have the abilitygiven the teachings of the description to modify the amino acid sequenceof a TCR, by maintaining the known anchor residues, and would be able todetermine whether such TCR variants maintain the ability to bind MHCclass I or II molecules/KVLEHVVRV (SEQ ID NO:1) complexes. The TCRvariants of the description retain the ability to bind MHC class I or IImolecules/KVLEHVVRV (SEQ ID NO:1) complexes. T-cells expressing the TCRvariants of the description can subsequently kill cells that express apolypeptide containing the natural amino acid sequence of the cognatepeptide, such as KVLEHVVRV (SEQ ID NO:1).

In an aspect, the peptides or TCRs disclosed herein can be modified bythe substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain of said peptide. For TCRs, preferably those substitutions arelocated at variable domains of TCR alpha chain and TCR beta chain. Suchsubstitutions may be of a conservative nature, for example, where oneamino acid is replaced by an amino acid of similar structure andcharacteristics, such as where a hydrophobic amino acid is replaced byanother hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

In an aspect, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of thedescription and yet still be encompassed by the disclosure herein. Inaddition, non-standard amino acids (i.e., other than the commonnaturally occurring proteinogenic amino acids) may also be used forsubstitution purposes to produce immunogens and immunogenic polypeptidesaccording to the present description.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would be simultaneously substituted.

As used herein, the term “murine” or “human,” when referring to anantigen recognizing construct, or a TCR, or any component of a TCRdescribed herein (e.g., complementarity determining region (CDR),variable region, constant region, α chain, and/or β chain), means a TCR(or component thereof), which is derived from a mouse or a humanunrearranged TCR locus, respectively.

T-Cell Receptors (TCRs)

In a preferred embodiment, the description relates a TCR comprising aTCR alpha chain shown in Table 2, and variants thereof; and a TCR betachain shown in Table 2, and variants thereof. In an aspect, a TCRdescribed herein has the ability to bind or specifically bind to amolecule of the human major histocompatibility complex (MHC)class-I/KVLEHVVRV (SEQ ID NO:1)/complex or to class II/KVLEHVVRV (SEQ IDNO:1)/complex.

TABLE 2 Representative TCRs according to present description TCR IDDescription Sequence R20P1H7 alpha chainMEKMLECAFIVLWLQLGWLSGEDQVTQSPEALRLQEG alpha ESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSA chainGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVQGENSGYSTLTFGKGTMLLVSPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMT LRLWSS (SEQ ID NO: 39)L segment MEKMLECAFIVLWLQLGWLSG (SEQ ID NO: 40) (TRAV20) V chainMEKMLECAFIVLWLQLGWLSGEDQVTQSPEALRLQEG (TRAV20)ESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVQ (SEQ ID NO: 41) CDR1VSGLRG (SEQ ID NO: 42) CDR2 LYS (SEQ ID NO: 43) CDR3CAVQGENSGYSTLTF (SEQ ID NO: 44) J segmentNSGYSTLTFGKGTMLLVSP (SEQ ID NO: 45) (TRAJ11) Constant DIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSK re-DSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACAN gion (TRAC)AFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 46) R20P1H7 beta chainMGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKL beta TVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVT chainDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSLGPGLAAYNEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALV LMAMVKRKDSRG(SEQ ID NO: 47)L segment MGPQLLGYVVLCLLGAGPL (SEQ ID NO: 48) (TRBV27) V chainMGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKL (TRBV27)TVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCAS SL (SEQ ID NO: 49) CDR1MNHEY (SEQ ID NO: 50) CDR2 SMNVEV (SEQ ID NO: 51) CDR3CASSLGPGLAAYNEQF (SEQ ID NO: 52) J chain YNEQFFGPGTRLTVL (SEQ ID NO: 53)(TRBJ2-1) constant re- EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPD gionHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCL (TRBC2)SSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 54) R7P1D5   alpha chainMKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGD al-SSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMD pha MKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEYS chainSASKIIFGSGTRLSIRPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLW SS (SEQ ID NO: 55) L segmentMKTFAGFSFLFLWLQLDCMSR (SEQ ID NO: 56) (TRAV5) V chainMKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGD (TRAV5)SSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAE (SEQ ID NO: 57) CDR1DSSSTY (SEQ ID NO: 58) CDR2 IFS (SEQ ID NO: 59) CDR3CAEYSSASKIIF ((SEQ ID NO: 60) J segmentYSSASKIIFGSGTRLSIRP (SEQ ID NO: 61) (TRAJ3) Constant re-NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSK gion (TRAC)DSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 62) R7P1D5 beta chainMGSWTLCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEV beta TLRCKPISGHDYLFWYRQTMMRGLELLIYFNNNVPIDDS chainGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASRANTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVK RKDSRG (SEQ ID NO: 63) L segmentMGSWTLCCVSLCILVAKHT (SEQ ID NO: 64) (TRBV12-4) V chainMGSWTLCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEV (TRBV12-4)TLRCKPISGHDYLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCAS (SEQ ID NO: 65) CDR1SGHDY (SEQ ID NO: 66) CDR2 FNNNVP (SEQ ID NO: 67) CDR3CASRANTGELFF (SEQ ID NO: 68) J chain NTGELFFGEGSRLTVL (SEQ ID NO: 69)(TRBJ2-1) constant re- EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPD gionHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCL (TRBC2)SSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 70) R10P2G12 alpha chainMLTASLLRAVIASICVVSSMAQKVTQAQTEISVVEKEDV alpha TLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQ chainNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSEGNSGNTPLVFGKGTRLSVIANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMT LRLWSS (SEQ ID NO: 71)L segment MLTASLLRAVIASICVVSSM (SEQ ID NO: 72) (TRAV19) V chainMLTASLLRAVIASICVVSSMAQKVTQAQTEISVVEKEDV (TRAV19)TLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALSE (SEQ ID NO:73) CDR1TRDTTYY (SEQ ID NO: 74) CDR2 RNSF (SEQ ID NO: 75) CDR3CALSEGNSGNTPLVF (SEQ ID NO: 76) J segmentNSGNTPLVFGKGTRLSVIA (SEQ ID NO: 77) (TRAJ29) Constant re-NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSK gion (TRAC)DSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 78) R10P2G12 beta chainMGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKV beta FLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKE chainKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSLSSGSHQETQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLM AMVKRKDSRG (SEQ ID NO: 79)L segment MGIRLLCRVAFCFLAVGLV(SEQ ID NO: 80) (TRBV28) V chainMGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKV (TRBV28)FLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSL (SEQ ID NO: 81) CDR1MDHEN (SEQ ID NO: 82) CDR2 SYDVKM (SEQ ID NO: 83) CDR3CASSLSSGSHQETQYF (SEQ ID NO: 84) J chain QETQYFGPGTRLLVL (SEQ ID NO: 85)(TRBJ2-5) constant re- EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPD gionHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCL (TRBC2)SSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 86)

The alpha and beta chains of alpha/beta TCR's, and the gamma and deltachains of gamma/delta TCRs, are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region (V), and joining region(J). The variable domain may also include a leader region (L). Beta anddelta chains may also include a diversity region (D). The alpha and betaconstant domains may also include C-terminal transmembrane (TM) domainsthat anchors the alpha and beta chains to the cell membrane.

In the present description, the term “TCR alpha variable domain”therefore refers to the concatenation of the TCR alpha V (TRAV) regionwithout leader region (L), and the TCR alpha J (TRAJ) region; and theterm “TCR alpha constant domain” refers to the extracellular TRACregion, or to a C-terminal truncated TRAC sequence, and optionally analpha transmembrane domain (VIGFRILLLKVAGFNLLMTL (SEQ ID NO:87)).

Likewise the term “TCR beta variable domain” refers to the concatenationof the TCR beta V (TRBV) region without leader region (L), and the TCRbeta D/J (TRBD/TRBJ) region; and the term TCR beta constant domainrefers to the extracellular TRBC region, or to a C-terminal truncatedTRBC sequence, and optionally a beta transmembrane domain(TILYEILLGKATLYAVLVSALVL (SEQ ID NO:88)).

With respect to gamma/delta TCRs, the term “TCR gamma variable domain”as used herein refers to the concatenation of the TCR gamma V (TRGV)without leader region (L), and TCR gamma J (TRGJ) regions; and the termTCR gamma constant domain refers to the extracellular TRGC region, or toa C-terminal truncated TRGC sequence. Likewise the term “TCR deltavariable domain” refers to the concatenation of the TCR delta V (TRDV)without leader region (L), and TCR delta D/J (TRDD/TRDJ) regions; andthe term TCR delta constant domain refers to the extracellular TRDCregion, or to a C-terminal truncated TRDC sequence.

In an embodiment, a TCR of the present description comprises or consistsof a TCR alpha chain at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical, preferably 90%, 95%, 96%, 97%, 98%, or99% identical, to a TCR alpha chain shown in Table 2. The TCR alphachains shown in Table 2 contain a leader (L) segment; a V chain; threecomplimentary determining regions (CDR1, CDR2 and CDR3); a joiningregion (J) and a constant region, as defined in Table 2.

In an embodiment, a TCR of the present description comprises or consistsof a TCR alpha variable domain at least 75%, 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical, preferably 90%, 95%, 96%,97%, 98%, or 99% identical, to a TCR alpha variable domain shown inTable 2.

In an embodiment, a TCR of the present description comprises or consistsof, a TCR alpha constant domain at least 75%, 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical, preferably 75% identical, toa TCR alpha constant domain shown in Table 2.

In an embodiment, a TCR of the present description comprises, orconsists of, a TCR alpha variable domain comprising at least one alphachain complementarity determining region (CDR) selected from the groupconsisting of an alpha chain CDR1, CDR2 and CDR3 shown in Table 2. In apreferred embodiment, the TCR alpha variable domain comprises an alphachain CDR3 shown in Table 2. In another preferred embodiment, the TCRalpha variable domain comprises an alpha chain CDR1, CDR2 and CDR3 shownin Table 2.

In a particularly preferred embodiment, a TCR of the present descriptioncomprises, or consists of, a TCR alpha variable domain having at least90% sequence identity to a TCR alpha variable domain of Table 2, andcomprises CDR1, CDR2 and CDR3 of the same alpha variable domain of Table2.

In an embodiment, a TCR of the present description comprises, orconsists of, a TCR beta chain at least 75%, 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical, preferably 90%, 95%, 96%,97%, 98%, or 99% identical, to a TCR beta chain shown in Table 2. TheTCR beta chains shown in Table 2 contain a leader (L) segment; a Vchain; three complimentary determining regions (CDR1, CDR2 and CDR3); ajoining region (J) and a constant region, as defined in Table 2.

In an embodiment, a TCR of the present description comprises, orconsists of, a TCR beta variable domain at least 75%, 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, preferably 90%,95%, 96%, 97%, 98%, or 99% identical, to a TCR beta variable domainshown in Table 2.

In an embodiment, a TCR of the present description comprises, orconsists of, a TCR beta constant domain at least 75%, 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, preferably 75%identical, to a TCR beta constant domain shown in Table 2.

In an embodiment, a TCR of the present description comprises, orconsists of, a TCR beta variable domain comprising at least one betachain complementarity determining region (CDR) selected from the groupconsisting of a beta chain CDR1, CDR2 and CDR3 shown in Table 2. In apreferred embodiment, the TCR beta variable domain comprises a betachain CDR3 shown in Table 2. In another preferred embodiment, the TCRbeta variable domain comprises a beta chain CDR1, CDR2 and CDR3 shown inTable 2.

In a particularly preferred embodiment, a TCR of the present descriptioncomprises, or consists of, a TCR beta variable domain having at least90% or 95% sequence identity to a TCR beta variable domain of Table 2,and comprises CDR1, CDR2 and CDR3 of the same TCR beta variable domainof Table 2.

The alpha chain variable domain may comprise one or more alpha CDRdomains having one, two, three or four amino acid substitutions relativeto the corresponding CDR sequence shown in Table 2. Likewise, the betachain variable domain may comprise one or more beta CDR domains havingone, two, three or four amino acid substitutions relative to thecorresponding beta CDR sequence shown in Table 2.

The TCR alpha chain and TCR beta chain may be fused to form a singlechain TCR. Alternatively, the TCR alpha and beta chains may be expressedas separate proteins which can be assembled into a heterodimer.

In one embodiment, any TCR alpha chain of Table 2 is paired with any TCRbeta chain of Table 2 to produce a TCR that specifically binds to aMAG-003 peptide-HLA molecule complex.

TCR R20P1H7

In one embodiment, a TCR of the present description comprises, orconsists of, the alpha chain and/or beta chain of TCR R20P1H7,corresponding to SEQ ID NOs:39 and 47, respectively.

The TCR alpha variable domain of TCR R20P1H7 comprises, or alternativelyconsists of, amino acids 22 to 133 of SEQ ID NO:39; the TCR alphaconstant domain of TCR R20P1H7 comprises, or alternatively consists of,amino acids 134-275 of SEQ ID NO:39; the TCR beta variable domain of TCRR20P1H7 comprises, or alternatively consists of, amino acids 20 to 135of SEQ ID NO:47; and the TCR beta constant domain comprises, oralternatively consists of, amino acids 136 to 315 of SEQ ID NO:47.

In a particular embodiment, a TCR of the present description comprises aTCR alpha chain at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical, preferably 90%, 95%, 96%, 97%, 98%, or 99%identical, to the TCR alpha chain of SEQ ID NO:39.

In another embodiment, a TCR of the present description comprises a TCRalpha variable domain at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical, preferably 90%, 95%, 96%, 97%, 98%, or99% identical, to the TCR alpha variable domain of SEQ ID NO:39.

In an embodiment, a TCR of the present description comprises a TCR alphaconstant domain at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical, preferably 75% identical, to the TCR alphaconstant domain of SEQ ID NO:39.

In an embodiment, a TCR of the present description comprises a TCR alphavariable domain comprising at least one alpha chain complementaritydetermining region (CDR) selected from the group consisting of the alphachain CDR1, CDR2 and CDR3 of SEQ ID NO:39. In a preferred embodiment,the TCR alpha variable domain comprises the alpha chain CDR3 of SEQ IDNO:39. In another preferred embodiment, the TCR alpha variable domaincomprises the alpha chain CDR1, CDR2 and CDR3 of SEQ ID NO:39.

In a particularly preferred embodiment, a TCR of the present descriptioncomprises a TCR alpha variable domain having at least 90% or 95%sequence identity to the TCR alpha variable domain of SEQ ID NO:39, andcomprises the CDR1, CDR2 and CDR3 of SEQ ID NO:39.

In another particular embodiment, a TCR of the present descriptioncomprises a TCR beta chain at least 75%, 80%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical, preferably 90%, 95%, 96%, 97%,98%, or 99% identical, to the TCR beta chain of SEQ ID NO:47.

In an embodiment, a TCR of the present description comprises a TCR betavariable domain at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical, preferably 90%, 95%, 96%, 97%, 98%, or 99%identical, to the TCR beta variable domain of SEQ ID NO:47.

In an embodiment, a TCR of the present description comprises a TCR betaconstant domain at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical, preferably 75% identical, to the TCR betaconstant domain of SEQ ID NO:47.

In an embodiment, a TCR of the present description comprises a TCR betavariable domain comprising at least one beta chain complementaritydetermining region (CDR) selected from the group consisting of the betachain CDR1, CDR2 and CDR3 of SEQ ID NO:47. In a preferred embodiment,the TCR beta variable domain comprises the beta chain CDR3 of SEQ IDNO:47. In another preferred embodiment, the TCR beta variable domaincomprises the beta chain CDR1, CDR2 and CDR3 of SEQ ID NO:47.

In a particularly preferred embodiment, a TCR of the present descriptioncomprises a TCR beta variable domain having at least 90% or 95% sequenceidentity to the TCR beta variable domain of SEQ ID NO:47, and comprisesCDR1, CDR2 and CDR3 of SEQ ID NO:47.

The alpha chain variable domain may comprise one or more alpha CDRdomains having one, two, three or four amino acid substitutions relativeto the corresponding CDR sequence of SEQ ID NO:39. Likewise, the betachain variable domain may comprise one or more beta CDR domains havingone, two, three or four amino acid substitutions relative to thecorresponding beta CDR sequence of SEQ ID NO:47.

TCR R7P1D5

In one embodiment, a TCR of the present description comprises, orconsists of, the alpha chain and/or beta chain of TCR R7P1 D5,corresponding to SEQ ID NOs:55 and 63, respectively.

The TCR alpha variable domain of TCR R7P1 D5 comprises, or alternativelyconsists of, amino acids 22 to 131 of SEQ ID NO:55; the TCR alphaconstant domain of TCR R7P1 D5 comprises, or alternatively consists of,amino acids 132 to 272 of SEQ ID NO:55; the TCR beta variable domain ofTCR R7P1 D5 comprises, or alternatively consists of, amino acids 20 to131 of SEQ ID NO:63; and the TCR beta constant domain comprises, oralternatively consists of, amino acids 132 to 310 of SEQ ID NO:63.

In a particular embodiment, a TCR of the present description comprises aTCR alpha chain at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical, preferably 90%, 95%, 96%, 97%, 98%, or 99%identical, to the TCR alpha chain of SEQ ID NO:55.

In another embodiment, a TCR of the present description comprises a TCRalpha variable domain at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical, preferably 90%, 95%, 96%, 97%, 98%, or99% identical, to the TCR alpha variable domain of SEQ ID NO:55.

In an embodiment, a TCR of the present description comprises a TCR alphaconstant domain at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical, preferably 75% identical, to the TCR alphaconstant domain of SEQ ID NO:55.

In an embodiment, a TCR of the present description comprises a TCR alphavariable domain comprising at least one alpha chain complementaritydetermining region (CDR) selected from the group consisting of the alphachain CDR1, CDR2 and CDR3 of SEQ ID NO:55. In a preferred embodiment,the TCR alpha variable domain comprises the alpha chain CDR3 of SEQ IDNO:55. In another preferred embodiment, the TCR alpha variable domaincomprises the alpha chain CDR1, CDR2 and CDR3 of SEQ ID NO:55.

In a particularly preferred embodiment, a TCR of the present descriptioncomprises a TCR alpha variable domain having at least 90% or 95%sequence identity to the TCR alpha variable domain of SEQ ID NO:55, andcomprises the CDR1, CDR2 and CDR3 of SEQ ID NO:55.

In another particular embodiment, a TCR of the present descriptioncomprises a TCR beta chain at least 75%, 80%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical, preferably 90% or 95% identical,to the TCR beta chain of SEQ ID NO:63.

In an embodiment, a TCR of the present description comprises a TCR betavariable domain at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical, preferably 90%, 95%, 96%, 97%, 98%, or 99%identical, to the TCR beta variable domain of SEQ ID NO:63.

In an embodiment, a TCR of the present description comprises a TCR betaconstant domain at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical, preferably 75% identical, to the TCR betaconstant domain of SEQ ID NO:63.

In an embodiment, a TCR of the present description comprises a TCR betavariable domain comprising at least one beta chain complementaritydetermining region (CDR) selected from the group consisting of the betachain CDR1, CDR2 and CDR3 of SEQ ID NO:63. In a preferred embodiment,the TCR beta variable domain comprises the beta chain CDR3 of SEQ IDNO:63. In another preferred embodiment, the TCR beta variable domaincomprises the beta chain CDR1, CDR2 and CDR3 of SEQ ID NO:63.

In a particularly preferred embodiment, a TCR of the present descriptioncomprises a TCR beta variable domain having at least 90% or 95% sequenceidentity to the TCR beta variable domain of SEQ ID NO:63, and comprisesCDR1, CDR2 and CDR3 of SEQ ID NO:63.

The alpha chain variable domain may comprise one or more alpha CDRdomains having one, two, three or four amino acid substitutions relativeto the corresponding CDR sequence of SEQ ID NO:55. Likewise, the betachain variable domain may comprise one or more beta CDR domains havingone, two, three or four amino acid substitutions relative to thecorresponding beta CDR sequence of SEQ ID NO:63.

TCR R10P2G12

In one embodiment, a TCR of the present description comprises, orconsists of, the alpha chain and/or beta chain of TCR R10P2G12,corresponding to SEQ ID NOs:71 and 79, respectively.

The TCR alpha variable domain of TCR R10P2G12 comprises, oralternatively consists of, amino acids 21 to 136 of SEQ ID NO:71; theTCR alpha constant domain of TCR R10P2G12 comprises, or alternativelyconsists of, amino acids 137 to 277 of SEQ ID NO:71; the TCR betavariable domain of TCR R10P2G12 comprises, or alternatively consists of,amino acids 20 to 134 of SEQ ID NO:79; and the TCR beta constant domaincomprises, or alternatively consists of, amino acids 135 to 313 of SEQID NO:79.

In a particular embodiment, a TCR of the present description comprises aTCR alpha chain at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical, preferably 90%, 95%, 96%, 97%, 98%, or 99%identical, to the TCR alpha chain of SEQ ID NO:71.

In another embodiment, a TCR of the present description comprises a TCRalpha variable domain at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identical, preferably 90%, 95%, 96%, 97%, 98%, or99% identical, to the TCR alpha variable domain of SEQ ID NO:71.

In an embodiment, a TCR of the present description comprises a TCR alphaconstant domain at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical, preferably 75% identical, to the TCR alphaconstant domain of SEQ ID NO:71.

In an embodiment, a TCR of the present description comprises a TCR alphavariable domain comprising at least one alpha chain complementaritydetermining region (CDR) selected from the group consisting of the alphachain CDR1, CDR2 and CDR3 of SEQ ID NO:71. In a preferred embodiment,the TCR alpha variable domain comprises the alpha chain CDR3 of SEQ IDNO:71. In another preferred embodiment, the TCR alpha variable domaincomprises the alpha chain CDR1, CDR2 and CDR3 of SEQ ID NO:71.

In a particularly preferred embodiment, a TCR of the present descriptioncomprises a TCR alpha variable domain having at least 90% or 95%sequence identity to the TCR alpha variable domain of SEQ ID NO:71, andcomprises the CDR1, CDR2 and CDR3 of SEQ ID NO:71.

In another particular embodiment, a TCR of the present descriptioncomprises a TCR beta chain at least 75%, 80%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical, preferably 90%, 95%, 96%, 97%,98%, or 99% identical, to the TCR beta chain of SEQ ID NO:79.

In an embodiment, a TCR of the present description comprises a TCR betavariable domain at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical, preferably 90%, 95%, 96%, 97%, 98%, or 99%identical, to the TCR beta variable domain of SEQ ID NO:79.

In an embodiment, a TCR of the present description comprises a TCR betaconstant domain at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical, preferably 75% identical, to the TCR betaconstant domain of SEQ ID NO:79.

In an embodiment, a TCR of the present description comprises a TCR betavariable domain comprising at least one beta chain complementaritydetermining region (CDR) selected from the group consisting of the betachain CDR1, CDR2 and CDR3 of SEQ ID NO:79. In a preferred embodiment,the TCR beta variable domain comprises the beta chain CDR3 of SEQ IDNO:79. In another preferred embodiment, the TCR beta variable domaincomprises the beta chain CDR1, CDR2 and CDR3 of SEQ ID NO:79.

In a particularly preferred embodiment, a TCR of the present descriptioncomprises a TCR beta variable domain having at least 90% or 95% sequenceidentity to the TCR beta variable domain of SEQ ID NO:79, and comprisesCDR1, CDR2 and CDR3 of SEQ ID NO:79.

The alpha chain variable domain may comprise one or more alpha CDRdomains having one, two, three or four amino acid substitutions relativeto the corresponding CDR sequence of SEQ ID NO:71. Likewise, the betachain variable domain may comprise one or more beta CDR domains havingone, two, three or four amino acid substitutions relative to thecorresponding beta CDR sequence of SEQ ID NO:79.

In a further preferred embodiment, a TCR of the present descriptionspecifically binds to a MAG-003 peptide-HLA molecule complex, whereinthe MAG-003 peptide is selected from KVLEHVVRV (SEQ ID NO:1) andvariants thereof, such as those shown in SEQ ID NO:2 to SEQ ID NO:24. Inan embodiment the HLA molecule is a class I MHC molecule selected fromthe group consisting of HLA-A, HLA-B, and HLA-C molecules. In oneembodiment the HLA molecule is HLA-A*02. In another embodiment, the HLAmolecule is a class II MHC molecule selected from the group consistingof HLA-DP, HLA-DQ, and HLA-DR.

TCRs of the present description preferably bind to a MAG-003 peptide-HLAmolecule complex with a binding affinity (K_(D)) of about 100 μM orless, about 50 μM or less, about 25 μM or less, or about 10 μM or less.More preferred are high affinity TCRs having binding affinities of about1 μM or less, about 100 nM or less, about 50 nM or less, about 25 nM orless. Nonlimiting examples of preferred binding affinity ranges for TCRsof the present description include about 1 nM to about 10 nM; about 10nM to about 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40nM; about 40 nM to about 50 nM; about 50 nM to about 60 nM; about 60 nMto about 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM;and about 90 nM to about 100 nM.

As used herein in connect with TCRs of the present description,“specific binding” and grammatical variants thereof are used to mean aTCR having a binding affinity (K_(D)) for a MAG-003 peptide-HLA moleculecomplex of 100 μM or less.

Alpha/beta heterodimeric TCRs of the present description may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned above,alpha/beta heterodimeric TCRs of the present description may have a TRACconstant domain sequence and a TRBC1 or TRBC2 constant domain sequence,and the TRAC constant domain sequence and the TRBC1 or TRBC2 constantdomain sequence of the TCR may be linked by the native disulfide bondbetween Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2.

TCRs of the present description may comprise a detectable label selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.TCRs of the present description may be conjugated to a therapeuticallyactive agent, such as a radionuclide, a chemotherapeutic agent, or atoxin.

In an embodiment, a TCR of the present description having at least onemutation in the alpha chain and/or having at least one mutation in thebeta chain has modified glycosylation compared to the unmutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCRalpha chain and/or TCR beta chain has a binding affinity for, and/or abinding half-life for, a MAG-003 peptide-HLA molecule complex, which isat least double that of a TCR comprising the unmutated TCR alpha chainand/or unmutated TCR beta chain. Affinity-enhancement of tumor-specificTCRs, and its exploitation, relies on the existence of a window foroptimal TCR affinities. The existence of such a window is based onobservations that TCRs specific for HLA-A2-restricted pathogens have KDvalues that are generally about 10-fold lower when compared to TCRsspecific for HLA-A2-restricted tumor-associated self-antigens (Aleksicet al. 2012; Kunert et al. 2013). It is now known, although tumorantigens have the potential to be immunogenic, because tumors arise fromthe individual's own cells only mutated proteins or proteins withaltered translational processing will be seen as foreign by the immunesystem. Antigens that are upregulated or overexpressed (so calledself-antigens) will not necessarily induce a functional immune responseagainst the tumor: T-cells expressing TCRs that are highly reactive tothese antigens will have been negatively selected within the thymus in aprocess known as central tolerance (Xing et al. 2012; Ruella et al.2014; Sharpe et al. 2015), meaning that only T-cells with low-affinityTCRs for self-antigens remain. Therefore, affinity of TCRs or variantsof the present description to MAG-003 have been enhanced by methods wellknown in the art as described below.

MAG-003 Peptides

The description provides a peptide comprising a sequence that isselected from the group of consisting of SEQ ID NO:1 to SEQ ID NO:24 ora variant thereof which is 88% homologous to SEQ ID NO:1 to SEQ IDNO:24, or a variant thereof that will induce T-cells cross-reacting witha peptide described herein. In an aspect, the peptides of thedescription have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or longer versions of a peptidedescribed herein to class II. In an aspect, a TCR described herein iscapable of binding to or specifically binding to a peptide describedherein.

In humans there are three different genetic loci that encode MHC class Imolecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

Table 3: Expression frequencies F of HLA-A*02 and HLA-A*24 and the mostfrequent HLA-DR serotypes. Frequencies are deduced from haplotypefrequencies Gf within the American population adapted from Mori et al.(Mori et al., 1997) employing the Hardy-Weinberg formula F=1−(1−Gf)².Combinations of A*02 or A*24 with certain HLA-DR alleles might beenriched or less frequent than expected from their single frequenciesdue to linkage disequilibrium. For details refer to Chanock et al.(Chanock et al., 2004).

TABLE 3 Calculated phenotype Allele Population from allele frequencyA*02 Caucasian (North America)  49.1% A*02 African American (NorthAmerica)  34.1% A*02 Asian American (North America)  43.2% A*02 LatinAmerican (North American)  48.3% DR1 Caucasian (North America)  19.4%DR2 Caucasian (North America)  28.2% DR3 Caucasian (North America) 20.6% DR4 Caucasian (North America)  30.7% DR5 Caucasian (NorthAmerica)  23.3% DR6 Caucasian (North America)  26.7% DR7 Caucasian(North America)  24.8% DR8 Caucasian (North America)   5.7% DR9Caucasian (North America)   2.1% DR1 African (North) American 13.20% DR2African (North) American 29.80% DR3 African (North) American 24.80% DR4African (North) American 11.10% DR5 African (North) American 31.10% DR6African (North) American 33.70% DR7 African (North) American 19.20% DR8African (North) American 12.10% DR9 African (North) American  5.80% DR1Asian (North) American  6.80% DR2 Asian (North) American 33.80% DR3Asian (North) American  9.20% DR4 Asian (North) American 28.60% DR5Asian (North) American 30.00% DR6 Asian (North) American 25.10% DR7Asian (North) American 13.40% DR8 Asian (North) American 12.70% DR9Asian (North) American 18.60% DR1 Latin (North) American 15.30% DR2Latin (North) American 21.20% DR3 Latin (North) American 15.20% DR4Latin (North) American 36.80% DR5 Latin (North) American 20.00% DR6Latin (North) American 31.10% DR7 Latin (North) American 20.20% DR8Latin (North) American 18.60% DR9 Latin (North) American  2.10% A*24Philippines   65% A*24 Russia Nenets   61% A*24:02 Japan   59% A*24Malaysia   58% A*24:02 Philippines   54% A*24 India   47% A*24 SouthKorea   40% A*24 Sri Lanka   37% A*24 China   32% A*24:02 India   29%A*24 Australia West   22% A*24 USA   22% A*24 Russia Samara   20% A*24South America   20% A*24 Europe   18%

MAGEA4 Gene

This gene is a member of the MAGEA gene family. The members of thisfamily encode proteins with 50 to 80% sequence identity to each other.The promoters and first exons of the MAGEA genes show considerablevariability, suggesting that the existence of this gene family enablesthe same function to be expressed under different transcriptionalcontrols. The MAGEA genes are clustered at chromosomal location Xq28.They have been implicated in some hereditary disorders, such asdyskeratosis congenita. At least four variants encoding the same proteinhave been found for this gene. (Provided by RefSeq, July 2008).

MAGEA4 localization has been described as cytoplasmic (Kim et al.,2015). However, MAGEA4 staining has also been detected in nuclei, withdifferential distribution between nucleus and cytoplasm inwell-differentiated versus less differentiated cancers (Sarcevic et al.,2003).

MAGEA4 is used as a male germ cell marker. It is not expressed ingonocytes, but expressed in pre-spermatogonia and mature germ cells(Mitchell et al., 2014).

TABLE 4 General cancer target Antigen properties EvaluationOver-expression in [cancer of interest] reported in literatureOver-expression in other cancers reported in literature + T-cellresponses against source protein-derived targets described + Oncofetalexpression pattern + Expression by cancer stem cells (−) Roles in cellcycle progression and tumor cell proliferation (−) Involvement in tumorinvasion, migration and metastasis Link to cancer-associated signalingpathways¹ Anti-apoptotic effects (−) Pro-angiogeniceffects/Neovascularisation Over-expression linked to poor prognosis incancer + Over-expression associated with advanced cancer stages +General cancer target Sub-cellular localization² CY Characterization ofsource protein in literature (−, +, ++, +++) + Cell type association³ TU¹TGF = Transforming growth factor; PI3K = Phosphatidylinositide3-kinases p53 = cellular tumor antigen p53; EGFR = epithelial growthfactor receptor; FGF2 = fibroblast growth factor 2; Wnt =Wnt/beta-catenin pathway (embryogenesis); Ras = Rat sarcomaproto-oncogene; NF-kB = Nuclear factor Kappa B (eukaryotic transcriptionfactor) ²CY = cytoplasmic; ³TU = tumor cellspMHC as Target

A phase I clinical trial investigated adoptive transfer ofTCR-engineered autologous CTLs reactive towards MAGEA4(143-151) bound toHLA-A*24:02 in esophageal cancer patients. Patients were givenTCR-transduced lymphocytes once, without preconditioning treatment,followed by subcutaneous immunizations with MAGEA4 peptide after 2 and 4weeks. No objective tumor regression was observed, possibly due to thelack of lymphodepleting regimen and administration of IL2 (Kageyama etal., 2015). Preclinical studies in mice had demonstrated thattransferred T-cells inhibited growth of MAGEA4-expressing tumor celllines inoculated in the mice, and that additional peptide vaccinationenhanced this antitumor activity (Shirakura et al., 2012).

Targeting MAGEA4 with adoptive CTL transfer is proposed as a treatmentoption of EBV-negative Hodgkin and non-Hodgkin lymphoma. Infused CTLstargeting EBV-derived peptides have been described to induce completeremissions in EBV(+) lymphoma patients. Therefore, targeting otherantigens expressed by lymphoma, including MAGEA4, is being explored as apossible treatment option (Cruz et al., 2011; Gerdemann et al., 2011).

Several studies have demonstrated the generation of MAGEA4 specificCD4(+) T-cells from healthy donors and cancer patients after incubationwith autologous antigen-presenting cells pulsed with overlapping peptidepools (Cesson et al., 2011; Gerdemann et al., 2011; Ohkuri et al.,2009).

MAG-003 peptide, i.e., KVLEHVVRV (SEQ ID NO:1), is aHLA-A*0201-restricted cytotoxic T lymphocyte (CTL) epitope of MAGEA4(amino acids 286-294). (Jia et al. 2010; Wu et al. 2011), the contentsof which are hereby incorporated by reference in their entirety. In anaspect, MAG-003 elicits peptide-specific CTLs both in vitro fromHLA-A*0201-positive PBMCs and in HLA-A*0201/Kb transgenic mice. Inanother aspect, the induced CTLs lyse target-cells in anHLA-A*0201-restricted fashion, demonstrating that MAG-003 isHLA-A*0201-restricted CTL epitope and serve as a target for therapeuticantitumoral vaccination (Jia et al. 2010), the content of which ishereby incorporated by reference in its entirety.

FIG. 1 shows MAG-003 peptide presentation in healthy tissues andcancers. The results are summarized in Table 5. Specifically, about4,000 and 2,000 copies of MAG-003 per cell are estimated in tumortissues from ovarian cancer (OC) and non-small cell lung cancer (NSCLC),respectively.

TABLE 5 MAG-003 presentation in healthy tissues and cancers. A*02Samples Mean intensity jScore Healthy 0 of 245 — — Cancer 14 of 3971.1e+07 0.000 HCC 1 of 16 2.9e+06 0.000 MEL 0 of 3 0.0e+00 OC 2 of 204.0e+07 0.000 pNSCLC 11 of 91 1.0e+07 0.000

Expression Profiling of Genes Encoding the Peptides of the Description

Over-presentation or specific presentation of TAAs on tumor cellscompared to healthy cells is sufficient for its usefulness inimmunotherapy, and some peptides are tumor-specific despite their sourceprotein occurring also in healthy tissues. Still, mRNA expressionprofiling adds an additional level of safety in selection of peptidetargets for immunotherapies. Especially for therapeutic options withhigh safety risks, such as affinity-matured TCRs, the ideal targetpeptide will be derived from a protein that is unique to the tumor andnot found on healthy tissues.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated aboveafter written informed consent had been obtained from each patient.Tumor tissue specimens were snapfrozen immediately after surgery andlater homogenized with mortar and pestle under liquid nitrogen. TotalRNA was prepared from these samples using TRI Reagent (Ambion,Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN, Hilden,Germany); both methods were performed according to the manufacturer'sprotocol.

RNAseq Experiments

Gene expression analysis of tumor and healthy tissue RNA samples wasperformed by next generation sequencing (RNAseq) by CeGaT (Tübingen,Germany). Sequencing libraries are prepared using the Illumina HiSeq v4reagent kit according to the provider's protocol (Illumina Inc, SanDiego, Calif., USA), which includes RNA fragmentation, cDNA conversionand addition of sequencing adaptors. Libraries derived from multiplesamples are mixed equimolarly and sequenced on the Illumina HiSeq 2500sequencer according to the manufacturer's instructions, generating 50 bpsingle end reads. Processed reads are mapped to the human genome(GRCh38) using the STAR software. Expression data are provided ontranscript level as RPKM (Reads Per Kilobase per Million mapped reads,generated by the software Cufflinks) and on exon level (total reads,generated by the software Bedtools), based on annotations of the ensemblsequence database (Ensembl77). Exon reads are normalized for exon lengthand alignment size to obtain RPKM values.

As shown in FIGS. 2-4, MAG-003 is highly expressed in cancer tissues andin low risk healthy tissues, such as testis, as compared with that ishigh risk and medium risk healthy tissues.

Tables 6-8 show RNASeq data (expression scores) of MAG-003 expression invarious cancers

TABLE 6 RNASeq Score 1 Tumor exonScore exonScore exonScore type tgScore(27242) (317034) (593984) BRCA 1.57 1.23 1.23 1.51 CRC 1.65 1.00 1.001.76 HCC 12.10 11.98 11.97 6.15 OC 56.60 18.45 18.44 57.74 OSCAR 58.423.49 3.49 60.40 PC 12.10 10.78 10.77 4.74 pGB 0.88 0.95 0.95 0.74 pNSCLC100.83 1.52 1.52 98.57 RCC 0.93 0.95 0.95 0.77 SCLC 56.41 28.32 28.30152.27

TABLE 7 RNASeq Score 3 Tumor exonScore exonScore exonScore type tgScore(27242) (317034) (593984) BRCA 7.48 5.11 5.11 6.01 CRC 8.35 1.05 1.057.90 HCC 123.03 210.33 210.30 42.22 OC 612.59 333.74 333.69 447.29 OSCAR632.95 47.41 47.40 468.45 PC 122.95 187.07 187.05 31.15 pGB 0.31 0.180.18 0.25 pNSCLC 1100.05 10.26 10.25 768.23 RCC 0.78 0.18 0.18 0.43 SCLC611.00 524.23 524.17 1190.36

TABLE 8 Tumor expression Exon- Exon- Exon- Tumor tumor40 tumor40 tumor40type tgtumor40 (27242) (317034) (593984) BRCA 0.12 0.04 0.04 0.17 CRC0.14 0.01 0.01 0.22 HCC 2.05 1.82 1.82 1.18 OC 11.19 3.16 3.16 13.72OSCAR 10.89 0.42 0.42 13.11 PC 2.09 1.65 1.65 0.89 pGB 0.00 0.00 0.000.01 pNSCLC 19.25 0.09 0.09 22.58 RCC 0.01 0.00 0.00 0.01 SCLC 10.184.53 4.53 33.35

In an aspect, a peptide consisting essentially of the amino acidsequence as indicated herein can have one or two non-anchor amino acids(see below regarding the anchor motif) exchanged without that theability to bind to a molecule of the human major histocompatibilitycomplex (MHC) class-I or -II is substantially changed or is negativelyaffected, when compared to the non-modified peptide. In anotherembodiment, in a peptide consisting essentially of the amino acidsequence as indicated herein, one or two amino acids can be exchangedwith their conservative exchange partners (see herein below) withoutthat the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II is substantiallychanged, or is negatively affected, when compared to the non-modifiedpeptide.

The amino acid residues that do not substantially contribute tointeractions with the TCR can be modified by replacement with otheramino acids whose incorporation do not substantially affect T-cellreactivity and does not eliminate binding to the relevant MHC. Thus,apart from the proviso given, the peptide of the description may be anypeptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 9 Variants and motif of the peptides Position 1 2 3 4 5 6 7 8 9SEQ ID NOs: 1-24 K V L E H V V R V Variants L A I L L L L A L I A A L AA A I Y L Y L L Y L A Y L I Y A Y A L Y A A Y A I Y Y L Y A Y I

Longer peptides may also be suitable in an aspect. It is possible thatMHC class I epitopes, although usually the actual epitope are residuesthat do not substantially affect proteolytic cleavage necessary toexpose the actual epitope during processing.

In an aspect, peptides of the description can be elongated by up to 1,2, 3, or 4 amino acids, that is 1, 2, 3 or 4 amino acids can be added toeither end of the peptide in any combination between 8 and 11 aminoacids long. It is preferred that the residues that flank between 4:0 and0:4. Combinations of the elongations according to the description can befound in Table 10.

TABLE 10 Combinations of the elongations of peptides of the descriptionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present description may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than 4 amino acids, preferably to a total length of up to30 amino acids. In an aspect, this elongation leads to MHC class IIbinding peptides. Binding to MHC class II can be tested by methods knownin the art.

Accordingly, the present description provides peptides and variants ofMHC class I epitopes, wherein the peptide or variant has an overalllength of between 8 and 100, preferably between 8 and 30, and mostpreferred between 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids,in case of the longer class II binding peptides the length can also be15, 16, 17, 18, 19, 20, 21 or 22 amino acids.

In an aspect, the peptide or variant according to the presentdescription will have the ability to bind to a molecule of the humanmajor histocompatibility complex (MHC) class I or II. Binding of apeptide or a variant to a MHC complex may be tested by methods known inthe art.

Preferably, when the T-cells specific for a peptide according to thepresent description are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 pM, andmost preferably no more than about 10 pM. It is also preferred that thesubstituted peptide be recognized by T-cells from more than oneindividual, at least two, and more preferably three individuals.

As used herein, the term “complex” refers to a molecule thatspecifically binds to an (e.g., antigenic) determinant. In oneembodiment, a complex is able to direct the entity to which it isattached (e.g., a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g., the complex of a peptide with MHC,according to the application at hand). In another embodiment a complexis able to activate signaling through its target antigen, for example aT-cell receptor complex antigen. Complexes include but are not limitedto antibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T-cells. To assess whether a molecule is acomplex binding to a target, binding assays can be performed.

“Specific” binding means that the complex (e.g. TCR) binds thepeptide-MHC-complex of interest better than other naturally occurringpeptide-MHC-complexes, to an extent that a complex armed with an activemolecule that is able to kill a cell bearing the specific target is notable to kill another cell without the specific target but presentingother peptide-MHC complex(es). Binding to other peptide-MHC complexes isirrelevant if the peptide of the cross-reactive peptide-MHC is notnaturally occurring, i.e., not derived from the human HLApeptidome.Tests to assess target-cell killing are well known in the art. Theyshould be performed using target-cells (primary cells or cell lines)with unaltered peptide-MHC presentation, or cells loaded with peptidessuch that naturally occurring peptide-MHC levels are reached.

Each complex can comprise a labelling which provides that the boundcomplex can be detected by determining the presence or absence of asignal provided by the label. For example, the complex can be labeledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualization of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each complex can be conjugated with a second active molecule such as forexample IL-21, anti-CD3, and anti-CD28.

Further information on polypeptide complexes may be found, for example,in the background section of WO 2014/071978A1, which is incorporated byreference in its entirety.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

Pharmaceutical compositions of the present description also include atleast one TCR, soluble TCR, nucleic acid, and/or host cell expressing aTCR of the present description, in a pharmaceutically acceptablecarrier.

Pharmaceutical compositions of the present description may also includepharmaceutically acceptable excipients and/or stabilizers.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Anextensive listing of excipients that can be used in such a composition,can be, for example, taken from A. Kibbe, Handbook of PharmaceuticalExcipients (Kibbe, 2000). The composition can be used for a prevention,prophylaxis and/or therapy of adenomateous or cancerous diseases.Exemplary formulations can be found in, for example, EP2112253, which isherein incorporated by reference in its entirety.

A further aspect of the description provides nucleic acids (for examplepolynucleotides) encoding a peptide, peptide variants, TCRs and TCRvariants of the description. The polynucleotide may be, for example,DNA, cDNA, PNA, RNA or combinations thereof, either single- and/ordouble-stranded, or native or stabilized forms of polynucleotides, suchas, for example, polynucleotides with a phosphorothioate backbone and itmay or may not contain introns so long as it codes for the peptide. Ofcourse, only peptides that contain naturally occurring amino acidresidues joined by naturally occurring peptide bonds are encodable by apolynucleotide. A still further aspect of the description provides anexpression vector capable of expressing a polypeptide according to thedescription.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of thedescription employs the polymerase chain reaction as disclosed by SaikiR K, et al. (Saiki et al., 1988). This method may be used forintroducing the DNA into a suitable vector, for example by engineeringin suitable restriction sites, or it may be used to modify the DNA inother useful ways as is known in the art. If viral vectors are used,pox- or adenovirus vectors are preferred.

In one aspect, to obtain T-cells expressing TCRs of the presentdescription, nucleic acids encoding TCR-alpha and/or TCR-beta chains ofthe present description are cloned into expression vectors, such asgamma retrovirus or lentivirus. The recombinant viruses are generatedand then tested for functionality, such as antigen specificity andfunctional avidity. An aliquot of the final product is then used totransduce the target T-cell population (generally purified from patientPBMCs), which is expanded before infusion into the patient.

In another aspect, to obtain T-cells expressing TCRs of the presentdescription, TCR RNAs are synthesized by techniques known in the art,e.g., in vitro transcription systems. The in vitro-synthesized TCR RNAsare then introduced into primary CD8+ T-cells obtained from healthydonors by electroporation to re-express tumor specific TCR-alpha and/orTCR-beta chains.

TCR chains introduced into a peripheral T-cell may compete withendogenous TCR chains for association with the CD3 complex, which isnecessary for TCR surface expression. Because a high level of TCRsurface expression is essential to confer appropriate sensitivity fortriggering by cells expressing the target tumor antigen (Cooper et al.,2000; Labrecque et al., 2001), strategies that enhance TCR-alpha andTCR-beta gene expression levels are an important consideration in TCRgene therapy.

To increase the expression, nucleic acids encoding TCRs of the presentdescription may be operably linked to strong promoters, such asretroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murinestem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin,ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter (Cooperet al., 2004; Jones et al., 2009), elongation factor (EF)-1a (Tsuji etal., 2005) and the spleen focus-forming virus (SFFV) promoter (Joseph etal., 2008). In a preferred embodiment, the promoter is heterologous tothe nucleic acid being expressed.

In addition to strong promoters, TCR expression cassettes of the presentdescription may contain additional elements that can enhance transgeneexpression, including a central polypurine tract (cPPT), which promotesthe nuclear translocation of lentiviral constructs (Follenzi et al.,2000), and the woodchuck hepatitis virus posttranscriptional regulatoryelement (wPRE), which increases the level of transgene expression byincreasing RNA stability (Zufferey et al., 1999).

The alpha and beta chains of a TCR of the present invention may beencoded by nucleic acids located in separate vectors, or may be encodedby polynucleotides located in the same vector.

Achieving high-level TCR surface expression requires that both theTCR-alpha and TCR-beta chains of the introduced TCR be transcribed athigh levels. To do so, the TCR-alpha and TCR-beta chains of the presentdescription may be cloned into bicistronic constructs in a singlevector, which has been shown to be capable of overcoming this obstacle.The use of a viral intraribosomal entry site (IRES) between theTCR-alpha and TCR-beta chains results in the coordinated expression ofboth chains, because the TCR-alpha and TCR-beta chains are generatedfrom a single transcript that is broken into two proteins duringtranslation, ensuring that an equal molar ratio of TCR-alpha andTCR-beta chains are produced. (Schmitt et al. 2009).

Nucleic acids encoding TCRs of the present description may be codonoptimized to increase expression from a host cell. Redundancy in thegenetic code allows some amino acids to be encoded by more than onecodon, but certain codons are less “optimal” than others because of therelative availability of matching tRNAs as well as other factors(Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta genesequences such that each amino acid is encoded by the optimal codon formammalian gene expression, as well as eliminating mRNA instabilitymotifs or cryptic splice sites, has been shown to significantly enhanceTCR-alpha and TCR-beta gene expression (Scholten et al., 2006).

Furthermore, mispairing between the introduced and endogenous TCR chainsmay result in the acquisition of specificities that pose a significantrisk for autoimmunity. For example, the formation of mixed TCR dimersmay reduce the number of CD3 molecules available to form properly pairedTCR complexes, and therefore can significantly decrease the functionalavidity of the cells expressing the introduced TCR (Kuball et al.,2007).

To reduce mispairing, the C-terminus domain of the introduced TCR chainsof the present description may be modified in order to promoteinterchain affinity, while decreasing the ability of the introducedchains to pair with the endogenous TCR. These strategies may includereplacing the human TCR-alpha and TCR-beta C-terminus domains with theirmurine counterparts (murinized C-terminus domain); generating a secondinterchain disulfide bond in the C-terminus domain by introducing asecond cysteine residue into both the TCR-alpha and TCR-beta chains ofthe introduced TCR (cysteine modification); swapping interactingresidues in the TCR-alpha and TCR-beta chain C-terminus domains(“knob-in-hole”); and fusing the variable domains of the TCR-alpha andTCR-beta chains directly to CD3ζ (CD3ζ fusion). (Schmitt et al. 2009).

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the description. Thus, the DNA encoding thepeptide or variant of the description may be used in accordance withknown techniques, appropriately modified in view of the teachingscontained herein, to construct an expression vector, which is then usedto transform an appropriate host cell for the expression and productionof the polypeptide of the description. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648, which are herein incorporated by reference intheir entirety.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the description may be joinedto a wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of thedescription are then cultured for a sufficient time and underappropriate conditions known to those skilled in the art in view of theteachings disclosed herein to permit the expression of the polypeptide,which can then be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.),plant-cells, animal cells and insect-cells. Preferably, the system canbe mammalian cells such as CHO cells available from the ATCC CellBiology Collection.

In an embodiment, a host cell is engineered to express a TCR of thepresent description. In preferred embodiments, the host cell is a humanT-cell or T-cell progenitor. In some embodiments the T-cell or T-cellprogenitor is obtained from a cancer patient. In other embodiments theT-cell or T-cell progenitor is obtained from a healthy donor. Host cellsof the present description can be allogeneic or autologous with respectto a patient to be treated. In one embodiment, the host is a gamma/deltaT-cell transformed to express an alpha/beta TCR.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (YIps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3×FLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent-cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of thedescription are encoded and thus expressed in a successive order(similar to “beads on a string” constructs). In doing so, the peptidesor peptide variants may be linked or fused together by stretches oflinker amino acids, such as for example LLLLLL, or may be linked withoutany additional peptide(s) between them. These constructs can also beused for cancer therapy, and may induce immune responses both involvingMHC I and MHC II.

The present description also relates to a host cell transformed with apolynucleotide vector construct of the present description. The hostcell can be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect-cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbás and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent description is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (Cohen et al.,1972) and (Green and Sambrook, 2012). Transformation of yeast-cells isdescribed in Sherman et al. (Sherman et al., 1986). The method of Beggs(Beggs, 1978) is also useful. With regard to vertebrate cells, reagentsuseful in transfecting such cells, for example calcium phosphate andDEAE-dextran or liposome formulations, are available from StratageneCloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877,USA. Electroporation is also useful for transforming and/or transfectingcells and is well known in the art for transforming yeast-cell,bacterial cells, insect-cells and vertebrate cells.

Successfully transformed cells, i.e., cells that contain a DNA constructof the present description, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the description areuseful in the preparation of the peptides of the description, forexample bacterial, yeast and insect-cells. However, other host cells maybe useful in certain therapeutic methods. For example,antigen-presenting cells, such as dendritic cells, may usefully be usedto express the peptides of the description such that they may be loadedinto appropriate MHC molecules. Thus, the current description provides ahost cell comprising a nucleic acid or an expression vector according tothe description.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the description provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment the TCRs, the nucleic acid or the expressionvector of the description are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g., between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantiallypure, or contained in a suitable vector or delivery system. The nucleicacid may be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g., Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Nonviral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T-cells for the respectiveopposite CDR as noted above.

The present description further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein which areherein incorporated by reference in their entirety) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable alternatives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. In anaspect, at least one or more aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides or the TCRs comprising, preferablyconsisting of, a sequence according to any of SEQ ID NO 25 to SEQ ID NO26, according to the description at hand with the MHC molecule, usingthe cell-SELEX (Systematic Evolution of Ligands by Exponentialenrichment) technique.

In one embodiment the description provides a method of producing a TCRas described herein, the method comprising culturing a host cell capableof expressing the TCR under conditions suitable to promote expression ofthe TCR.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*02-negative healthy donors withA2/MAG-003 monomers, incubating the PBMCs with tetramer-phycoerythrin(PE) and isolating the high avidity T-cells by fluorescence activatedcell sorting (FACS)-Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*02-negative healthy donors withA2/p286-1Y2L monomers, incubating the PBMCs with tetramer-phycoerythrin(PE) and isolating the high avidity T-cells by fluorescence activatedcell sorting (FACS)-Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*02-negative healthy donors withA2/p286-1Y2L9L monomers, incubating the PBMCs withtetramer-phycoerythrin (PE) and isolating the high avidity T-cells byfluorescence activated cell sorting (FACS)-Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRαβ geneloci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with MAG-003, incubating PBMCs obtained from the transgenic micewith tetramer-phycoerythrin (PE), and isolating the high avidity T-cellsby fluorescence activated cell sorting (FACS)-Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRαβ geneloci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with p286-1Y2L, incubating PBMCs obtained from the transgenic micewith tetramer-phycoerythrin (PE), and isolating the high avidity T-cellsby fluorescence activated cell sorting (FACS)-Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRαβ geneloci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with p286-1Y2L9L, incubating PBMCs obtained from the transgenicmice with tetramer-phycoerythrin (PE), and isolating the high avidityT-cells by fluorescence activated cell sorting (FACS)-Calibur analysis.

The present description further relates to the method according to thedescription, wherein the T-cell comprises an expression vector capableof expressing A TCR according to the present description.

The present description further relates to a method of killingtarget-cells in a patient which target-cells aberrantly express MAG-003,the method comprising administering to the patient an effective numberof TCRs, soluble TCRs and/or T-cells as according to the presentdescription.

The present description further relates to the use of any TCR described,a nucleic acid according to the present description, an expressionvector according to the present description, a cell according to thepresent description, or an activated cytotoxic T lymphocyte according tothe present description as a medicament or in the manufacture of amedicament. The present description further relates to a use accordingto the present description, wherein the medicament is active againstcancer.

The present description further relates to a use according to thedescription, wherein said cancer cells are non-small cell lung cancercells or other solid or haematological tumor cells such as non-smallcell lung cancer, small cell lung cancer, renal cell cancer, braincancer, gastric cancer, colorectal cancer, hepatocellular cancer,pancreatic cancer, prostate cancer, leukemia, breast cancer, Merkel cellcarcinoma, melanoma, ovarian cancer, urinary bladder cancer, uterinecancer, gallbladder and bile duct cancer and esophageal cancer.

The present description further relates to a method of killing cancercells comprising contact the cancer cells with a host cell of thepresent description. In one embodiment, the host cell expresses a TCR ofthe present description. In one embodiment the host cell is a T-cell orT-cell progenitor. In one embodiment, In a preferred embodiment thecancer cells are selected from non-small cell lung cancer cells or othersolid or haematological tumor cells such as non-small cell lung cancer,small cell lung cancer, renal cell cancer, brain cancer, gastric cancer,colorectal cancer, hepatocellular cancer, pancreatic cancer, prostatecancer, leukemia, breast cancer, Merkel cell carcinoma, melanoma,ovarian cancer, urinary bladder cancer, uterine cancer, gallbladder andbile duct cancer and esophageal cancer. In some embodiments, the TCR isconjugated to a therapeutically active agent. In certain embodiments thetherapeutically active agent is selected from the group consisting of aradionuclide, a chemotherapeutic agent, and a toxin.

The present invention further relates to a method of treating cancercomprising administering to a subject in need thereof a host cell of thepresent invention. In one embodiment, the host cell expresses a TCR ofthe present description. In one embodiment the host cell is a T-cell orT-cell progenitor. In one embodiment the host cell is autologous to thesubject to be treated. In another embodiment the host cell is allogeneicto the subject to be treated. In a preferred embodiment the cancer cellsare selected from non-small cell lung cancer cells or other solid orhaematological tumor cells such as non-small cell lung cancer, smallcell lung cancer, renal cell cancer, brain cancer, gastric cancer,colorectal cancer, hepatocellular cancer, pancreatic cancer, prostatecancer, leukemia, breast cancer, Merkel cell carcinoma, melanoma,ovarian cancer, urinary bladder cancer, uterine cancer, gallbladder andbile duct cancer and esophageal cancer.

In some embodiments, the TCR is conjugated to a therapeutically activeagent. As used herein, the term “therapeutically active agent” means acompound used to treat or prevent a disease or undesirable medicalcondition. In one embodiment, the therapeutically active agent is usedto treat or prevent cancer. In certain embodiments the therapeuticallyactive agent is selected from the group consisting of a radionuclide, achemotherapeutic agent, and a toxin.

TCRs, nucleic acids and host cells of the present description, andpharmaceutical compositions thereof, may be administered to a subject inneed thereof by routes known in the art, and may vary depending on thetype of cancer to be treated. Routes of administration include, forexample, local administration (such as intratumoral) and parenteraladministration such as subcutaneous, intraperitoneal, intramuscular,intravenous, intraportal and intrahepatic. In a preferred embodiment,TCRs, nucleic acids or host cells of the present description, orpharmaceutical compositions thereof, are administered to a subject bylocal infusion, for example using an infusion pump and/or cathetersystem, to a site to be treated, such as a solid tumor. In oneembodiment, a composition of the present description is infused into asolid tumor, a blood vessel that feeds a solid tumor, and/or the areasurrounding a solid tumor.

In preferred embodiments, compositions of the present description areadministered to a subject using a dosing regimen of at least twoadministrations separated by at least 24 hours. Dosing regimens suitablefor administering compositions of the present description include, forexample, once a day, once every two days, and once every three days.More preferred dosing regimens include once a week, twice a week, onceevery other week, once a month, and twice a month. In particularembodiments, a dose escalation regimen is used, wherein a series ofincreasing doses is administered to a subject over a period of days,weeks or months.

Effective doses of host cells expressing TCRs of the present inventioninclude, for example at least about 10⁴, at least about 10⁵, at leastabout 10⁶, at least about 10′, at least about 10⁸, at least about 10⁹,and at least 10¹⁰ host cells per dose. In one embodiment, host cells ofthe present description are administered in a dose of between about 10⁴to about 10¹⁰ cells per dose, preferably in a dose of between about 10⁵to about 10⁹ cells per dose. In preferred embodiments, doses areadministered in a dosing regimen over the course of at least two or moredosing cycles.

The present invention also relates to a method of treating cancercomprising administering a TCR, a nucleic acid, or a host cell of thepresent description in combination with at least one chemotherapeuticagent and/or radiation therapy.

Also provided is a method of treating cancer in a subject in needthereof, comprising:

a) isolating a cell from said subject;b) transforming the cell with at least one vector encoding a TCR of thepresent description to produce a transformed cell;c) expanding the transformed cell to produce a plurality of transformedcells; andd) administering the plurality of transformed cells to said subject.

Also provided is a method of treating cancer in a subject in needthereof, comprising:

a) isolating a cell from a healthy donor;b) transforming the cell with a vector encoding a TCR of the presentdescription to produce a transformed cell;c) expanding the transformed cell to produce a plurality of transformedcells; andd) administering the plurality of transformed cells to said subject.

Also provided is a method of detecting cancer in a biological samplecomprising:

a) contacting the biological sample with a TCR of the presentdescription;b) detecting binding of the TCR to the biological sample.

In some embodiments the method of detecting cancer is carried out invitro, in vivo or in situ.

The present description further relates to particular marker proteinsand biomarkers based on the peptides according to the presentdescription, herein called “targets” that can be used in the diagnosisand/or prognosis of non-small cell lung cancer. The present descriptionalso relates to the use of these novel targets for cancer treatment.

It is a further aspect of the description to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g., by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target-cells. In another aspect, it is expressedin T-cells used for adoptive transfer. See, for example, WO2004/033685A1, WO 2004/074322A1, and WO 2013/057586A1, the contents ofwhich are incorporated by reference in their entirety.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present description can be used to verify apathologist's diagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody or TCR is labeled with a radionucleotide (suchas ¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than1×100.

Antibodies or TCRs for diagnostic use may be labeled with probessuitable for detection by various imaging methods. Methods for detectionof probes include, but are not limited to, fluorescence, light, confocaland electron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesand/or TCRs may be directly or indirectly labeled with said probes.Attachment of probes to the antibodies and/or TCRs includes covalentattachment of the probe, incorporation of the probe into the antibody orTCR, and the covalent attachment of a chelating compound for binding ofprobe, amongst others well recognized in the art. Forimmunohistochemistry, the disease tissue sample may be fresh or frozenor may be embedded in paraffin and fixed with a preservative such asformalin. The fixed or embedded section contains the sample arecontacted with a labeled primary antibody and secondary antibody,wherein the antibody is used to detect the expression of the proteins insitu.

The invention further pertains to the following items:

Item 1. A TCR comprising an alpha chain and a beta chain, wherein thealpha chain comprises a TCR alpha variable domain at least 90% identicalto the amino acid sequence of any of SEQ ID NO:39, SEQ ID NO:55 and SEQID NO:71; and the beta chain comprises a TCR beta variable domain atleast 90% identical to any of SEQ ID NO:47, SEQ ID NO:63 and SEQ IDNO:79, and wherein the TCR specifically binds to a MAG-003 peptide-MHCmolecule complex.

Item 2. The TCR of item 1, further comprising a TCR alpha constantdomain and a TCR beta constant domain, wherein the TCR alpha constantdomain is at least 70% identical to a TCR alpha constant domain of anyof SEQ ID NO:39, SEQ ID NO:55 and SEQ ID NO:71, and the beta constantdomain is at least 70% identical to a TCR beta constant domain of any ofSEQ ID NO:47, SEQ ID NO:63 and SEQ ID NO:79.

Item 3. The TCR of any of items 1 or 2, wherein the alpha constantdomain comprises the alpha transmembrane domain VIGFRILLLKVAGFNLLMTL(SEQ ID NO:97) and the beta constant domain comprises the betatransmembrane domain TILYEILLGKATLYAVLVSALVL (SEQ ID NO:88).

Item 4. The TCR of any of items 1 to 3, wherein the TCR alpha variabledomain consists of the amino acid sequence of SEQ ID NO:39; and the TCRbeta variable domain consists of the amino acid sequence of SEQ IDNO:47.

Item 5. The TCR of any of items 1 to 3, wherein the TCR alpha variabledomain consists of the amino acid sequence of SEQ ID NO:55; and the TCRbeta variable domain consists of the amino acid sequence of SEQ IDNO:63.

Item 6. The TCR of any of items 1 to 3, wherein the TCR alpha variabledomain consists of the amino acid sequence of SEQ ID NO:71; and the TCRbeta variable domain consists of the amino acid sequence of SEQ IDNO:79.

Item 7. The TCR of any of items 1 to 6, wherein the TCR alpha constantdomain consists of the TCR alpha constant domain of SEQ ID NO:39, andthe TCR beta constant domain consists of the TCR beta constant domain ofSEQ ID NO:47.

Item 8. The TCR of any of items 1 to 6, wherein the TCR alpha constantdomain consists of the TCR alpha constant domain of SEQ ID NO:55, andthe TCR beta constant domain consists of the TCR beta constant domain ofSEQ ID NO:63.

Item 9. The TCR of any of items 1 to 6, wherein the TCR alpha constantdomain consists of the TCR alpha constant domain of SEQ ID NO:71, andthe TCR beta constant domain consists of the TCR beta constant domain ofSEQ ID NO:79.

Item 10. The TCR of any of items 1 to 9, comprising an alpha chainconsisting of SEQ ID NO:39 and a beta chain consisting of SEQ ID NO:47.

Item 11. The TCR of any of items 1 to 9, comprising an alpha chainconsisting of SEQ ID NO:55 and a beta chain consisting of SEQ ID NO:63.

Item 12. The TCR of any of items 1 to 9, comprising an alpha chainconsisting of SEQ ID NO:71 and a beta chain consisting of SEQ ID NO:79.

Item 13. The TCR of any of items 1 to 12, wherein the TCR alpha chaincomprises at least one alpha chain complementarity determining region(CDR) selected from the group consisting of an alpha chain CDR1, CDR2and CDR3 of SEQ ID NO:39, SEQ ID NO:55 and SEQ ID NO:71; and/or the TCRbeta chain comprises at least one beta chain complementarity determiningregion (CDR) selected from the group consisting of a beta chain CDR1,CDR2 and CDR3 of SEQ ID NO:47, SEQ ID NO:63 and SEQ ID NO:79.

Item 14. The TCR of any of items 1 to 13, wherein the TCR alpha chaincomprises all three CDRs of SEQ ID NO:39, SEQ ID NO:55 or SEQ ID NO:71.

Item 15. The TCR of any of items 1 to 13, wherein the TCR beta chaincomprises all three CDRs of SEQ ID NO:47, SEQ ID NO:63 or SEQ ID NO:79.

Item 16. The TCR of any of items 1 to 15, wherein the alpha chain andbeta chain are fused to form a single chain TCR.

Item 17. The TCR of any of items 1 to 16, wherein the alpha and/or betachain comprises a detectable label.

Item 18. The TCR of item 17, wherein the detectable label is selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.

Item 19. The TCR of any of items 1 to 18, wherein the alpha and/or betachain is conjugated to a therapeutically active agent.

Item 20. The TCR of item 19, wherein the therapeutically active agent isselected from the group consisting of a radionuclide, a chemotherapeuticagent and a toxin.

Item 21. A TCR comprising a gamma chain and a delta chain, wherein thegamma chain comprises at least one complementarity determining region(CDR) selected from the group consisting of an alpha chain CDR1, CDR2and CDR3 of SEQ ID NO:39, SEQ ID NO:55 and SEQ ID NO:71; and/or the TCRdelta chain comprises at least one complementarity determining region(CDR) selected from the group consisting of a beta chain CDR1, CDR2 andCDR3 of SEQ ID NO:47, SEQ ID NO:63 and SEQ ID NO:79, and wherein the TCRspecifically binds to a MAG-003 peptide-MHC molecule complex.

Item 22. The TCR of any of items 1 to 21, wherein the TCR specificallybinds to a MAG-003 peptide-MHC molecule complex, wherein the MAG-003peptide is selected from the group consisting of SEQ ID NOs:1-24, andthe MHC molecule is an HLA class I or HLA class II molecule.

Item 23. A nucleic acid encoding the alpha chain and/or beta chain ofthe TCR of any of items 1 to 20, or the gamma chain and/or delta chainof the TCR of item 21.

Item 24. An expression vector comprising the nucleic acid of item 23operably linked to at least one promoter sequence.

Item 25. A host cell transformed with the expression vector of item 24.

Item 26. The host cell of item 25 which is a T cell or T cellprogenitor.

Item 27. The host cell of item 26 wherein the T cell or T cellprogenitor is obtained from a cancer patient.

Item 28. The host cell of item 26 wherein the T cell or T cellprogenitor is obtained from a healthy donor.

Item 29. A pharmaceutical composition comprising the TCR of any of items1 to 22, a nucleic acid of item 23, an expression vector of item 24,and/or a host cell of any one of items 25 to 28; and a pharmaceuticallyacceptable carrier, and optionally, pharmaceutically acceptableexcipients and/or stabilizers.

Item 30. A method for producing a TCR that specifically binds to thepeptide of SEQ ID NO:1 when presented by an MHC molecule, said methodcomprising culturing the host cell of any one of items 25 to 28 underconditions suitable to promote expression of the TCR.

Item 31. A method of treating cancer comprising administering to asubject in need thereof the TCR of any of items 1 to 22, the nucleicacid of item 23, the expression vector of item 24, the host cell of anyone of items 25 to 28, and/or the pharmaceutical composition of item 29.

Item 32. The method of item 31, wherein the TCR is expressed on thesurface of a host cell.

Item 33. The method of item 31, wherein the host cell is selected fromthe group consisting of a T cell or T cell progenitor.

Item 34. The method of item 33, wherein the T cell or T cell progenitoris autologous.

Item 35. The method of item 33, wherein the T cell or T cell progenitoris allogeneic.

Item 36. The method of item 31, wherein the TCR is conjugated to atherapeutically active agent.

Item 37. The method of item 36, wherein the therapeutically active agentis selected from the group consisting of a radionuclide, achemotherapeutic agent and a toxin.

Item 38. The method of any of items 31 to 37, wherein the cancer isnon-small cell lung cancer, small cell lung cancer, renal cell cancer,brain cancer, gastric cancer, colorectal cancer, hepatocellular cancer,pancreatic cancer, prostate cancer, leukemia, breast cancer, Merkel cellcarcinoma, melanoma, ovarian cancer, urinary bladder cancer, uterinecancer, gallbladder and bile duct cancer, esophageal cancer, or acombination thereof.

Item 39. The method of any of items 31 to 38, further comprisingadministering to the subject at least one chemotherapeutic agent.

Item 40. The method of any of items 31 to 39, further comprisingadministering radiation therapy to the subject.

Item 41. A method of treating cancer in a subject in need thereof,comprising:

a) isolating a cell from said subject;b) transforming the cell with a vector encoding the TCR of any of items1 to 22 to produce a transformed cell;c) expanding the transformed cell to produce a plurality of transformedcells; andd) administering the plurality of transformed cells to said subject.

Item 42. The method of item 41, wherein the cell is selected from a Tcell or a T cell progenitor.

Item 43. A method of treating cancer in a subject in need thereof,comprising:

a) isolating a cell from a healthy donor;b) transforming the cell with a vector encoding the TCR of any of items1 to 22 to produce a transformed cell;c) expanding the transformed cell to produce a plurality of transformedcells; andd) administering the plurality of transformed cells to said subject.

Item 44. The method of item 43, wherein the cell is selected from a Tcell or a T cell progenitor.

Item 45. A method of detecting cancer in a biological sample comprising:

a) contacting the biological sample with the TCR of any of items 1 to22, andb) detecting binding of the TCR to the biological sample.

Item 46. The method of item 45, wherein the TCR comprises a detectablelabel.

Item 47. The method of item 46, wherein the detectable label is selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.

Item 48. The method of any of items 45 to 47, wherein said detecting iscarried out in vitro, in vivo or in situ.

Item 49. The method of any of items 45 to 48, wherein said cancer isnon-small cell lung cancer, small cell lung cancer, renal cell cancer,brain cancer, gastric cancer, colorectal cancer, hepatocellular cancer,pancreatic cancer, prostate cancer, leukemia, breast cancer, Merkel cellcarcinoma, melanoma, ovarian cancer, urinary bladder cancer, uterinecancer, gallbladder and bile duct cancer, esophageal cancer, or acombination thereof.

Item 50. The host cell of any of items 25 to 28 wherein the T cell is agamma/delta T cell.

Item 51. A method of killing target-cells in a patient whichtarget-cells aberrantly express MAG-003, the method comprisingadministering to the patient an effective number of T-cells expressing aTCR of any of items 1 to 22, the nucleic acid of item 23, the expressionvector of item 24, the host cell of any one of items 25 to 28, and/orthe pharmaceutical composition of item 29.

Item 52. The TCR of any of items 1 to 22 which is a soluble TCR.

Item 53. The TCR of any of items 1 to 22, wherein the alpha chaincomprises a TCR alpha variable domain at least 95% identical to theamino acid sequence of any of SEQ ID NO:39, SEQ ID NO:55 and SEQ IDNO:71; and the beta chain comprises a TCR beta variable domain at least95% identical to any of SEQ ID NO:47, SEQ ID NO:63 and SEQ ID NO:79, andwherein the TCR specifically binds to a MAG-003 peptide-MHC moleculecomplex.

Item 54. The TCR of any of items 1 to 22 having at least one mutation inthe alpha chain relative to SEQ ID NO:39, SEQ ID NO:55 or SEQ ID NO:71and/or having at least one mutation in the beta chain relative to SEQ IDNO:47, SEQ ID NO:63 and SEQ ID NO:79, and wherein the TCR has a bindingaffinity for, and/or a binding half-life for, a MAG-003 peptide-HLAmolecule complex, which is at least double that of the unmutated TCR forthe same peptide.

Item 55. The TCR of any of items 1 to 22 having at least one mutation inthe alpha chain relative to SEQ ID NO:39, SEQ ID NO:55 or SEQ ID NO:71and/or having at least one mutation in the beta chain relative to SEQ IDNO:47, SEQ ID NO:63 and SEQ ID NO:79, and wherein the TCR has modifiedglycosylation compared to the unmutated TCR.

Item 56. The method of any of items 31 to 44 or 51, wherein the TCR ofany of items 1 to 22, the nucleic acid of item 23, the expression vectorof item 24, the host cell of any of items 25 to 28 or the pharmaceuticalcomposition of item 29 is administered in at least two administrationsseparated by at least 24 hours.

Item 57. The method of item 56, wherein the TCR of any of items 1 to 22,the nucleic acid of item 23, the expression vector of item 24, the hostcell of any of items 25 to 28 or the pharmaceutical composition of item29 is administered to the subject over a period of days, weeks ormonths.

Item 58. The method of items 56 or 57, wherein the TCR of any of items 1to 22, the nucleic acid of item 23, the expression vector of item 24,the host cell of any of items 25 to 28 or the pharmaceutical compositionof item 29 is administered by local infusion.

Item 59. The method of any of item 58, wherein the local infusion isadministered by an infusion pump and/or a catheter system.

Item 60. The method of items 58 or 59, wherein said local infusion isinto a solid tumor, a blood vessel that feeds a solid tumor, and/or thearea surrounding a solid tumor.

Item 61. The method of any of items 31 to 44, 51 or 56 to 60, whereinthe TCR of any of items 1 to 22, the nucleic acid of item 23, theexpression vector of item 24, the host cell of any of items 25 to 28 orthe pharmaceutical composition of item 29 is administered in a dose ofabout 10⁴ to about 10¹⁰ cells per dose.

Item 62. A TCR comprising at least one alpha chain complementaritydetermining region (CDR) selected from the group consisting of an alphachain CDR1, CDR2 and CDR3 of SEQ ID NO:39, SEQ ID NO:55 and SEQ IDNO:71; and/or at least one beta chain complementarity determining region(CDR) selected from the group consisting of a beta chain CDR1, CDR2 andCDR3 of SEQ ID NO:47, SEQ ID NO:63 and SEQ ID NO:79, and wherein the TCRspecifically binds to a MAG-003 peptide-MHC molecule complex.

EXAMPLES

Allo-reactive settings can be used to circumvent self-tolerance andyield T-cells with a higher avidity when compared to T-cells derivedfrom autologous settings, i.e., patients. Examples of such settingsinclude in vitro generation of allo-HLA reactive, peptide-specificT-cells (Sadovnikova et al. 1998; Savage et al. 2004; Wilde et al.2012), and immunization of mice transgenic for human-MHC or human TCR(Stanislawski et al. 2001; Li et al. 2010).

Example 1: In Vitro Generation of Allo-HLA Reactive, Peptide-SpecificT-Cells (Savage et al. 2004)

PBMCs from HLA-A*02-positive and HLA-A*02-negative healthy donors wereused after obtaining informed consent. Recombinant biotinylated HLA-A2class I monomers and A2 fluorescent tetramers containing MAG-003 wereobtained from MBLI (Woburn, Mass.). PBMCs were incubated withanti-CD20SA diluted in phosphate buffered saline (PBS) for 1 hour atroom temperature, washed, and incubated with the biotinylated A2/MAG-003monomers for 30 minutes at room temperature, washed, and plated at 3×10⁶cells/well in 24-well plates in RPMI with 10% human AB serum.Interleukin 7 (IL-7; R&D Systems, Minneapolis, Minn.) was added on day 1at 10 ng/mL and IL-2 (Chiron, Harefield, United Kingdom) was added at 10U/mL on day 4. Over a 5-week period cells were restimulated weekly withfresh PBMCs, mixed with responder cells at a 1:1 ratio, and plated at3×10⁶/well in 24-well plates.

To obtain high avidity T-cells, approximately 10⁶ PBMCs withHLA-A2/MAG-003 tetramer-phycoerythrin (PE) (obtained from MBLI) wereincubated for 30 minutes at 37° C., followed by anti-CD8-fluoresceinisothiocyanate (FITC)/allophycocyanin (APC) for 20 minutes at 4° C.,followed by fluorescence activated cell sorting (FACS)-Calibur analysis.Sorting was done with a FACS-Vantage (Becton Dickinson, Cowley, Oxford,United Kingdom). Sorted tetramerpositive cells were expanded in 24-wellplates using, per well, 2×10⁶ sorted cells, 2×10⁶ irradiated A2-negativePBMCs as feeders, 2×10⁴ CD3/CD28 beads/mL (Dynal, Oslo Norway), and IL-2(1000 U/mL). The high avidity T-cells, thus obtained, were then used toidentify and isolate TCRs using techniques known in the art, such assingle cell 5′ RACE (Rapid Amplification of cDNA Ends). Non-redundantTCR DNAs were then analyzed for amino acid/DNA sequences determinationand cloning into expression vectors using methods well known in the art.

Example 2: Immunization of Mice Transgenic for Human-MHC or Human TCR

MAG-003 are used to immunize transgenic mice with the entire human TCRαβgene loci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency. (Li et al. 2010).To obtain high avidity T-cells, PBMCs obtained from the transgenic miceare incubated with tetramer-phycoerythrin (PE) followed by cell sortingas described above. The high avidity T-cells, thus obtained, are thenused to identify and isolate TCRs for amino acid/DNA sequencesdetermination and cloning into expression vectors using methodsdescribed in the art.

In an aspect, MAG-003 and its variants, i.e., p286-1Y2L (having 2 aminoacid substitutions, SEQ ID NO:13) and p286-1Y2L9L (having 3 amino acidsubstitutions, SEQ ID NO:14) exhibit potent binding affinity andstability towards HLA-A*0201 molecule. In particular, p286-1Y2L9L showedthe capability to induce specific CTLs which, in an aspect, lyse thetarget cancer cells from both PBMCs of healthy donors and HLA-A2.1/Kbtransgenic mice. See, for example, (Wu et al. 2011), the content ofwhich is hereby incorporated by reference in its entirety.

To obtain high avidity TCRs for MHC I or II/p286-1Y2L or p286-1Y2L9Lcomplexes, these peptides can be used in methods described in Examples 1and 2. The high avidity T-cells, thus obtained, are then used toidentify and isolate TCRs for amino acid/DNA sequences determination andcloning into expression vectors using methods described are in the art.

Example 3: Cloning of TCRs

Methods of cloning TCRs are known in the art, for example, as describedin U.S. Pat. No. 8,519,100, which is hereby incorporated by reference inits entirety for said methods. The alpha chain variable region sequencespecific oligonucleotide A1 (ggaattccatatgagtcaacaaggagaagaagatcc SEQ IDNO:26) which encodes the restriction site NdeI, an introduced methioninefor efficient initiation of expression in bacteria, and an alpha chainconstant region sequence specific oligonucleotide A2(ttgtcagtcgacttagagtctctcagctggtacacg SEQ ID NO:27) which encodes therestriction site SalI are used to amplify the alpha chain variableregion. In the case of the beta chain, a beta chain variable regionsequence specific oligonucleotide B1(tctctcatatggatggtggaattactcaatccccaa SEQ ID NO:28) which encodes therestriction site NdeI, an introduced methionine for efficient initiationof expression in bacteria, and a beta chain constant region sequencespecific oligonucleotide B2 (tagaaaccggtggccaggcacaccagtgtggc SEQ IDNO:29) which encodes the restriction site AgeI are used to amplify thebeta chain variable region.

The DNA sequences encoding the TCR alpha chain cut with NdeI and SalIare ligated into pGMT7+Cα vector, which was cut with NdeI and XhoI. TheDNA sequences encoding the TCR beta chain cut with NdeI and AgeI wasligated into separate pGMT7+Cβ vector, which was also cut with NdeI andAgeI. Ligated plasmids are transformed into competent Escherichia colistrain XL1-blue cells and plated out on LB/agar plates containing 100μg/ml ampicillin. Following incubation overnight at 37° C., singlecolonies are picked and grown in 10 ml LB containing 100 μg/mlampicillin overnight at 37° C. with shaking. Cloned plasmids arepurified using a Miniprep kit (Qiagen) and the insert is sequenced usingan automated DNA sequencer (Lark Technologies).

Three TCRs (R20P1H7, R7P1 D5 and R10P2G12, see table 2), each encodingtumor specific TCR-alpha and TCR-beta chains, were isolated andamplified from T-cells of healthy donors. Cells from healthy donors werein vitro stimulated according to the method described in Walter et. al.2003. Target-specific cells were single-cell sorted usingtarget-specific multimers for subsequent TCR isolation. TCR sequenceswere isolated via 5′ RACE by standard methods as described by e.g.Molecular Cloning a laboratory manual fourth edition by Green andSambrook. All three TCRs were derived from HLA-A2 positive donors. Thealpha and beta variable regions of TCRs R20P1H7, R7P1 D5 and R10P2G12were sequenced.

The R20P1H7 TCR alpha variable domain was found to have an amino acidsequence corresponding to residues 22-133 of SEQ ID NO:39. The R20P1H7TCR beta variable domain was found to have an amino acid sequencecorresponding to residues 20-135 of SEQ ID NO:47.

The R7P1 D5 TCR alpha variable domain has an amino acid sequencecorresponding to residues 22-131 of SEQ ID NO:55. The R7P1 D5 TCR betavariable domain has an amino acid sequence corresponding to residues20-131 of SEQ ID NO:63.

The R10P2G12 TCR alpha variable domain has an amino acid sequencecorresponding to residues 21-136 of SEQ ID NO:71. The R10P2G12 TCR betavariable domain has an amino acid sequence corresponding to residues20-134 of SEQ ID NO:79.

Phage display can be used to generate libraries of TCR variants toidentify high affinity mutants. The TCR phage display and screeningmethods described in (Li et al, (2005) Nature Biotech 23 (3): 349-354)can be applied to a reference TCR selected from the TCRs described inTable 2.

For example, all three CDR regions of the alpha chain sequence of SEQ IDNOs: 39, 55 and 71; and all three CDR regions of the beta chain sequenceof SEQ ID NOs: 47, 55 and 79 can be targeted by mutagenesis, and eachCDR library panned and screened separately.

Accordingly, TCRs with affinities and/or binding half-lives at leasttwice that of a reference TCR (and therefore impliedly at least twicethat of the native TCR) are identified.

TCR heterodimers are refolded using the method including the introducedcysteines in the constant regions to provide the artificial inter-chaindisulphide bond. In that way TCRs are prepared, consisting of (a) thereference TCR beta chain, together with mutated alpha chains; (b) thereference TCR alpha chain together with mutated beta chains; and (c)various combinations of beta and alpha chains including the mutantvariable domains.

The interaction between high affinity soluble disulfide-linked TCRs, andTCR variants, and the native peptide KVLEHVVRV (SEQ ID NO:1) HLA-A*02complex can be analysed using the BIAcore method.

High avidity TCR variants can also be selected from a library of CDRmutants by yeast, or T-cell display (Holler et al. 2003; Chervin et al.2008). Candidate TCR variants, thus, provide guidance to designmutations of the TCR's CDRs to obtain high avidity TCR variants (Robbinset al. 2008; Zoete et al. 2007).

Example 4: Autologous T-Cell Engineering

T-cells can be engineered to express high avidity TCRs (so-called TCRtherapies) or protein-fusion derived chimeric antigen receptors (CARs)that have enhanced antigen specificity to MHC I/MAG-003 complex or MHCII/MAG-003 complex. In an aspect, this approach overcomes some of thelimitations associated with central and peripheral tolerance, andgenerate T-cells that will be more efficient at targeting tumors withoutthe requirement for de novo T-cell activation in the patient.

In one aspect, to obtain T-cells expressing TCRs of the presentdescription, nucleic acids encoding the tumor specific TCR-alpha and/orTCR-beta chains identified and isolated, as described in Examples 1-3,are cloned into expression vectors, such as gamma retrovirus orlentivirus. The recombinant viruses are generated and then tested forfunctionality, such as antigen specificity and functional avidity. Analiquot of the final product is then used to transduce the target T-cellpopulation (generally purified from patient PBMCs), which is expandedbefore infusion into the patient.

In another aspect, to obtain T-cells expressing TCRs of the presentdescription, TCR RNAs were synthesized by techniques described in theart, e.g., in vitro transcription systems. The in vitro-synthesized TCRRNAs were then introduced into primary CD8+ T-cells obtained fromhealthy donors by electroporation to re-express tumor specific TCR-alphaand/or TCR-beta chains.

To determine specificity and affinity of TCRs, the transformed CD8+T-cells were co-incubated with MAG-003-loaded target cells or withtarget cells loaded with homologous but unrelated peptide RABGAP1L-001(SEQ ID NO:91), AXIN1-001 (SEQ ID NO:92), ANO5-001 (SEQ ID NO:93),TPX2-001 (SEQ ID NO:94), SYNE3-001 (SEQ ID NO:95), MIA3-001 (SEQ IDNO:96), HERC4-001 (SEQ ID NO:97), PSME2-001 (SEQ ID NO:98), HEATR5A-001(SEQ ID NO:99) or CNOT1-003 (SEQ ID NO:100) or control peptideNYESO1-001 (SEQ ID NO:101), followed by IFN-γ release assay. Unloadedtarget cells and CD8+ T-cells alone served as controls. IFN-γ secretionfrom CD8+ T-cells is indicative of T-cell activation with cytotoxicactivity.

TABLE 11 % MAG-003 % NYESO1- Donor/ TET-positive 001 TET- HLA- primarypositive pri- TCR A2 IFNγ CD8+ T- mary CD8+ No. TCR Code (+ or −)(pg/ml) EC50 cells T-cells 1 R7P1D5 HBC- 400-  ~3 nM 7.99 0.87 583/(+)1050 2 R20P1H7 HBC- 200-900 ~10 nM 5.73 0.87 689/(+) 3 R10P2G12 HBC-200- −10 nM 5.38 0.87 673/(+) 1500

As shown in FIGS. 5-7 all primary CD8+ T-cells transformed with TCRs ofthe present disclosure, after co-incubation with MAG-003-loaded targetcells, released much higher levels of IFN-γ than that stimulated byunrelated peptide-loaded target cells, and the controls. Target peptidetitration analysis showed EC50 ranges from about 3 nM to about 10 nM(Table 11). These results suggest that TCRs of the present invention canactivate cytotoxic T-cell activity, e.g., IFN-γ release, throughspecific interaction with the MHC/MAG-003 complex.

T-cell activation was further confirmed using a tetramer stainingtechnique to detect MHC/MAG-003-binding activated cytotoxic T-cells. Asshown in FIG. 8 and Table 11, a higher percentage of TCR-expressing CD8+T-cells were stained positive fluorescent-labeled MHC/MAG-003 tetramerthan that with MHC/NYESO1-001 control tetramer or mock control. As acontrol, primary CD8+ T-cells transformed with TCRs, such as 1G4 TCR,which is known to bind specifically to MHC/NYESO1-001 complex, wasreadily activated by NYESO1-001-loaded target cells. The alpha and betachains of TCR 1G4 are shown in SEQ ID NO:89 and 90, respectively:

1G4 alpha chain (SEQ ID NO: 89):METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGSYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 1G4 beta chain (SEQ ID NO: 90):MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVL MAMVKRKDSRG

To determine the binding motif of the TCRs for the MAG-003/MHC complex,positional alanine scanning analysis was performed at each of the 9amino acids of the MAG-003 peptide. Alanine-substituted MAG-003 peptidesare shown in Table 12.

TABLE 12 Position: 1 2 3 4 5 6 7 8 9 MAG-003 (SEQ ID NO: 1) K V L E H VV R V MAG-003 A1 (SEQ ID NO: 30) A V L E H V V R V MAG-003 A2 (SEQ IDNO: 31) K A L E H V V R V MAG-003 A3 (SEQ ID NO: 32) K V A E H V V R VMAG-003 A4 (SEQ ID NO: 33) K V L A H V V R V MAG-003 A5 (SEQ ID NO: 34)K V L E A V V R V MAG-003 A6 (SEQ ID NO: 35) K V L E H A V R V MAG-003A7 (SEQ ID NO: 36) K V L E H V A R V MAG-003 A8 (SEQ ID NO: 37) K V L EH V V A V MAG-003 A9 (SEQ ID NO: 38) K V L E H V V R A

CD8+ T-cells electroporated with RNA encoding TCR of interest wereco-incubated with target cells loaded with MAG-003, MAG-003-A1 toMAG-003-A9, or an unrelated NYESO1-001 peptide, followed by IFNγ releaseassay, as described above.

Results of positional alanine scanning analysis are shown in FIGS. 9-11and summarized in Table 13.

TABLE 13 TCR MAG-003 positions enable TCR binding R7P1D5 1, 3, 5, 7, 8R20P1H7 1, 3, 4, 5, 7, 8 R10P2G12 1, 2, 3, 4, 5, 8

A genome-wide screen for A*02-binding peptides with an identical motifrevealed no potentially cross-reactive peptides for the TCRs R20P1H7,R7P1 D5 and R10P2G12, respectively. These results suggest that the TCRsdescribed herein exhibit a very specific recognition pattern with areduced risk of off-target effects.

To determine efficacy of T-cells expressing TCRs described herein,primary CD8+ T-cells electroporated with RNA of the TCRs R7P1 D5,R20P1H7 and R10P2G12 were co-incubated with different human cancer celllines, e.g., A-375 (human melanoma cell line, which is positive forHLA-A2 and MAG-003 expression), T98G (human glioblastoma cell line,which is HLA-A2-positive and MAG-003-negative), and SK-BR-3 (humanbreast cancer cell line, which is HLA-A2-negative and MAG-003-negative),followed by IFNγ release assay.

As shown in FIG. 12A-C, IFNγ release was observed in A-375 cells, whichare HLA-A2-positive and MAG-003-positive, but not in T98G and SK-BR-3cells, which have basal levels of IFNγ release that is comparable tothat of effector cell only control. In addition, CD8+ T-cellstransformed with TCR R7P1 D5, but not controls, also exhibited increasedIFNγ release when co-incubated with the human non-small cell cancer cellline, H1755 (data not shown). These results indicate that T-cellsexpressing TCRs R7P1 D5, R20P1H7 and R10P2G12 can specifically inducecytotoxic activity targeting cancer cells in a HLA-A2/MAG-003 specificmanner.

The present description provides TCRs that are useful in treatingcancers/tumors, preferably melanoma and non-small cell lung cancer thatover- or exclusively present MAG-003.

Example 5: Allogeneic T-Cell Engineering

Gamma delta (γδ) T cells, which are non-conventional T lymphocyteeffectors implicated in the first line of defense against pathogens, caninteract with and eradicate tumor cells in a MHC-independent mannerthrough activating receptors, among others, TCR-gamma and TCR-deltachains. These γδ T cells display a preactivated phenotype that allowsrapid cytokine production (IFN-γ, TNF-α) and strong cytotoxic responseupon activation. These T-cells have anti-tumor activity against manycancers and suggest that γδ T cell-mediated immunotherapy is feasibleand can induce objective tumor responses. (Braza et al. 2013).

Recent advances using immobilized antigens, agonistic monoclonalantibodies (mAbs), tumor-derived artificial antigen presenting cells(aAPC), or combinations of activating mAbs and aAPC have been successfulin expanding gamma delta T-cells with oligoclonal or polyclonal TCRrepertoires. For example, immobilized major histocompatibility complexClass-I chain-related A was a stimulus for γδ T-cells expressing TCRδ1isotypes, and plate-bound activating antibodies have expanded Vδ1 andVδ2 cells ex vivo. Clinically sufficient quantities of TCRδ1, TCRδ2, andTCRδ1^(neg)TCRδ2^(neg) have been produced following co-culture on aAPC,and these subsets displayed differences in memory phenotype andreactivity to tumors in vitro and in vivo. (Deniger et al. 2014).

In addition, γδ T-cells are amenable to genetic modification asevidenced by introduction of TCR-alpha and TCR-beta chains. (Hiasa etal. 2009). Another aspect of the present description relates toproduction of γδ T-cells expressing TCR-alpha and TCR-beta that bind toMAG-003. To do so, γδ T-cells are expanded by methods described byDeniger et al. 2014, followed by transducing the recombinant virusesexpressing the TCRs that bind to MAG-003 (as described in Example 3)into the expanded γδ T-cells. The virus-transduced γδ T-cells are theninfused into the patient.

Example 6: Safety of MAG-003-Specific TCRs

Three MAG-003-specific TCRs (R7P1 D5, R10P2G12, R20P1H7, see Table 2),each encoding tumor specific TCR-alpha and TCR-beta chains, wereisolated and amplified from T-cells of healthy donors. Cells fromhealthy donors were in vitro stimulated according to a method previouslydescribed (Walter et al., 2003 J Immunol., November 15; 171(10):4974-8)and target-specific cells were single-cell sorted using HLA-A*02multimers and then used for subsequent TCR isolation. TCR sequences wereisolated via 5′ RACE by standard methods as described by e.g. MolecularCloning a laboratory manual fourth edition by Green and Sambrook. Thealpha and beta variable regions of TCRs R7P1D5, R10P2G12 and R20P1H7were sequenced and cloned for further functional characterization. TCRsR7P1 D5, R10P2G12 and R20P1H7 are derived from a HLA-A*02 positivedonor.

TABLE 13 Target Cell Types Cell type Abbrevation source Normal HumanDermal Fibroblasts NHDF Primary cells Human Coronary Artery SmoothHCASMC Primary cells Muscle Cells Human Bronchial Smooth Muscle CellsHBSMC Primary cells Human Renal Epithelial Cells HREpC Primary cellsHuman Cardiomyocytes HCM Primary cells Human Cardiac Microvascular HCMECPrimary cells Endothelial Cells Human Hepatocytes HH Primary cells HumanAstrocytes HA Primary cells Human Perineural Cells HPC Primary cellsHuman Meningeal Cells HMC Primary cells Human Neurons HN iPSC-derivedcells

TCRs of interest were expressed in human T cells via RNA electroporationand the T cells were assessed for IFN-γ release after co-culture withdifferent target cells, such as T2 cells loaded with different peptidesas well as tumor cell lines and primary cells from healthy tissues.T-cell activation data are shown in absolute IFN-γ levels or anormalized way as indicated below.

Efficacy of CD8+ T cells expressing TCRs R7P1 D5, R10P2G12 and R20P1H7was determined by T cell activation studies (IFN-γ release) usingdifferent tumor cell lines as target cells. The characterization of thesafety profile of TCRs of interest was approached by testing thepotential activation of TCR-expressing T cells upon co-culture withisolated primary cell types from healthy tissues and induced pluripotentstem cell (iPSC)-derived cell types from HLA-A*02-positive donors (Table13). Cell types were selected in a manner to cover most of the criticalorgans like e.g. brain, heart and liver and different cell types asepithelium, endothelium or smooth muscle. Tumor cell lines were analyzedside-by-side as positive and negative controls.

Normalization method for IFNγ release:

Mean_(norm (TCRoi; co))=[mean_((TcRoi; co))−mean_((TCRoi; effector only))]−[mean_((mock; co))−mean_((mock; effector only))]

The respective CV_(norm) was calculated:

CV _(norm (TCRoi; co))=[CV _((TCRoi; co)) ² +CV_((TCRoi; effector only)) ² +CV _((mock;co)) ² +CV_((mock; effector only)) ²]{circumflex over ( )}[½]

TCRoi=effector cells expressing TCR of interestMock=effector cells without exogenous TCR expressionCo=effector cells co-cultured with target cellsEffector only=effector cells not co-culturedMean(norm)=mean IFNγ release (normalized)CV(norm)=coefficient of variation (normalized)

Results

For CD8+ T cells expressing TCR R7P1 D5, R10P2G12 or R20P1H7, noactivation was observed upon co-culture with HLA-A*02 positive celltypes from healthy tissues (see FIG. 13), while there was a activitytowards the tumor cell lines A-375 and NCI-H1755 expressing HLA-A*02 andMAGEA4 as source gene for MAG-003 peptide. A similar pattern ofreactivity was observed with CD8+ T cells expressing the NYESO1-specificTCR 1G4, which were found to be reactive towards NYESO1 expressingHLA-A*02 positive tumor cell lines but not towards the indicated panelof healthy tissue cells.

T-cell activation upon co-culture with cell lines expressing HLA-A*02and MAGEA4 reflects the recognition of endogenously expressed andprocessed target pHLA by TCRs R7P1D5, R10P2G12 and R20P1H7.

The safety analysis indicates the absence of un-expectedcross-reactivity or potential alloreactivity of TCR R7P1 D5, R10P2G12and R20P1H7 towards healthy tissue primary cells. This is noteworthy asthe tested primary cell types represent thousands of normal HLA ligandsin the context of HLA-A*02 and likely also represent additionalallogeneic HLA alleles that vary from normal cell type to normal celltype.

Example 7: Lentiviral Expression of TCR of the Invention

T cell products were generated starting from bulk total Peripheral BloodMononuclear Cells (PBMCs) isolated from healthy donors following a smallscale ACTengine manufacturing process. The lentiviral vector backboneexpressing R7P1 D5 TCR of the invention for manufacturing ACTengine Tcells was designed based on previous studies and multiple clinicaltrials (Porter et al., 2011; Kalos et al., 2011; and others) Briefly,thawed and rested PBMCs were activated with CD3/CD28 antibodies andtransduced with concentrated viral supernatants manufactured usingvarious lentiviral vectors carrying R7P1 D5 TCR transgene with differentorientations of α and β chains and various combinations of promotors andenhancer sequences (constructs were denoted as R71-R78). Two of the 8constructs (R74, R75) were eliminated due to low titers and/or poorproductivity. Cells transduced with the remaining 6 viral supernatantswere expanded and evaluated for transgene expression by flow cytometry.

Orientations were as follows: Alpha-beta: R71, R72, 75, 76; beta-alpha:R73, R74, R77, 78, IG4.

All T cell products showed comparable viability, percentage oflymphocytes as well as CD3+ T-cells. Percentage of CD4+ and CD8+ T-cellsvaried among the donors. No differences were observed in terms oftoxicity among all 6 viral supernatants. R7P1 D5 TCR lentiviralconstruct R73 consistently induced higher TCR expression as detected onthe surface by tetramer binding. Based on transgene expressionlentiviral constructs follow the order: R73>R78>R77>R71>R76>R72 (FIG.14).

Additional experiments with 2 new donors were done to confirm thetransducibility of the R7P1 D5 TCR R73 virus. Transduced cell from allexperiments were tested for functional evaluation in cytokine releaseand killing assays. In all donors tested, R73 transduced T cells derivedfrom whole PBMCs of four donors exhibited significantly higher IFNγproduction upon co-culturing with A375 cells expressing MAGE-A4 antigenas compared to non-transduced T cells (FIG. 15A). IFNγ response wasstrictly antigen specific as induction in presence of MCF-7 cellslacking MAGE-A4 expression was below background levels. In addition,EC₅₀ value derived from T2 titration curves representing IFNγ quantitiesdetected in response to decreasing concentration of MAG-003 peptide was10.0 nM in both donors (FIG. 15B).

In addition to cytokine release, R7P1 D5 TCR (R73 virus) transduced Tcell products were tested in a 4 hour Cr51 release killing assay.Recognition and lysis of MAGE-A4+A375 tumor cells presentingendogenously processed MAG003MAG-003 peptide were evalu-ated. LV-R73transduced T cells displayed increased killing over NTnon-transducedcells at all E:T ratios (FIG. 16A, B). Cytotoxic effects seen with R73transduced cells were me-diated in antigen specific manner as percentagelysis observed with MAGE-A4+ targets was significantly higher thanobserved with MAGE-MAGEA-A4-negative target cells (FIG. 16C).

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1. A method of treating a patient who has cancer that presents a peptideconsisting of the amino acid sequence of KVLEHVVRV (SEQ ID NO: 1) in acomplex with HLA-A*02, comprising administering to the patient apopulation of transformed CD8+ T cells expressing at least one vectorencoding a T cell receptor (TCR), wherein the TCR comprises SEQ ID NO:58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 66, SEQ ID NO: 67, and SEQID NO: 68, wherein the TCR is capable of binding to a peptide consistingof the amino acid sequence of KVLEHVVRV (SEQ ID NO: 1) in a complex withHLA-A*02, and wherein the cancer is selected from non-small cell lungcancer (NSCLC), melanoma, and ovarian cancer (OC).
 2. The method ofclaim 1, wherein the population of transformed cells are produced by amethod comprising isolating a cell from a subject, transforming the cellwith at least one vector encoding the TCR to produce a transformed cell,and expanding the transformed cell to produce the population oftransformed cells.
 3. The method of claim 2, wherein the subject is thepatient.
 4. The method of claim 2, wherein the subject is a healthydonor.
 5. The method of claim 1, wherein the TCR comprises an α chaincomprising the amino acid sequence of SEQ ID NO: 55 and a β chaincomprising the amino acid sequence of SEQ ID NO:
 63. 6. The method ofclaim 1, wherein the population of transformed cells are administered inthe form of a pharmaceutical composition.
 7. The method of claim 8,wherein the pharmaceutical composition comprises a chemotherapeuticagent selected from the group consisting of asparaginase, busulfan,carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil,gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab,vinblastine, and vincristine.
 8. The method of claim 1, wherein the TCRcomprises: a CDR1α chain comprising the amino acid sequence of SEQ IDNO: 58, a CDR2α chain comprising the amino acid sequence of SEQ ID NO:59, a CDR3α chain comprising the amino acid sequence of SEQ ID NO:
 60. aCDR1β chain comprising the amino acid sequences of SEQ ID NO: 66, aCDR2β chain comprising the amino acid sequence of SEQ ID NO: 67, and aCDR3β chain comprising the amino acid sequence of SEQ ID NO:
 68. 9. Themethod of claim 1, wherein the TCR comprises a CDR1α chain consisting ofthe amino acid sequence of SEQ ID NO: 58, a CDR2α chain comprising theamino acid sequence of SEQ ID NO: 59, a CDR3α chain consisting of theamino acid sequence of SEQ ID NO: 60, a CDR1β chain consisting of theamino acid sequence of SEQ ID NO: 66, a CDR2β chain comprising the aminoacid sequence of SEQ ID NO: 67, and a CDR3β chain consisting of theamino acid sequence of SEQ ID NO:
 68. 10. The method of claim 1, whereinthe TCR comprises a CDR1α chain consisting of the amino acid sequence ofSEQ ID NO: 58, a CDR2α chain consisting of the amino acid sequence ofSEQ ID NO: 59, a CDR3α chain comprising the amino acid sequence of SEQID NO: 60, a CDR1β chain consisting of the amino acid sequence of SEQ IDNO: 66, a CDR2β chain consisting of the amino acid sequence of SEQ IDNO: 67, and a CDR3β chain comprising the amino acid sequence of SEQ IDNO:
 68. 11. The method of claim 1, wherein the TCR comprises a CDR1αchain consisting of the amino acid sequence of SEQ ID NO: 58, a CDR2αchain consisting of the amino acid sequence of SEQ ID NO: 59, a CDR3αchain consisting of the amino acid sequence of SEQ ID NO: 60, a CDR1βchain consisting of the amino acid sequence of SEQ ID NO: 66, a CDR2βchain consisting of the amino acid sequence of SEQ ID NO: 67, and aCDR3β chain consisting of the amino acid sequence of SEQ ID NO:
 78. 12.The method of claim 1, wherein the cancer is ovarian cancer.
 13. Themethod of claim 1, wherein the cancer is non-small cell lung cancer. 14.The method of claim 1, wherein the binding affinity (K_(D)) for the TCRbinding to a peptide consisting of the amino acid sequence of KVLEHVVRV(SEQ ID NO: 1) in a complex with an MHC class I molecule is about 100 μMor less.
 15. The method of claim 11, wherein the binding affinity(K_(D)) for the TCR binding to a peptide consisting of the amino acidsequence of KVLEHVVRV (SEQ ID NO: 1) in a complex with an MHC class Imolecule is about 100 μM or less.
 16. The method of claim 1, wherein theTCR comprises a CDR1α chain comprising the amino acid sequence of SEQ IDNO: 58, a CDR2α chain consisting of the amino acid sequence of SEQ IDNO: 59, a CDR3α chain consisting of the amino acid sequence of SEQ IDNO: 60, a CDR1β chain comprising the amino acid sequence of SEQ ID NO:66, a CDR2β chain consisting of the amino acid sequence of SEQ ID NO:67, and a CDR3β chain consisting of the amino acid sequence of SEQ IDNO:
 68. 17. The method of claim 1, wherein the TCR comprises a CDR1αchain comprising the amino acid sequence of SEQ ID NO: 58, a CDR2α chaincomprising the amino acid sequence of SEQ ID NO: 59, a CDR3α chainconsisting of the amino acid sequence of SEQ ID NO: 60, a CDR1β chaincomprising the amino acid sequence of SEQ ID NO: 66, a CDR2β chaincomprising the amino acid sequence of SEQ ID NO: 67, and a CDR3β chainconsisting of the amino acid sequence of SEQ ID NO:
 68. 18. The methodof claim 1, wherein the TCR comprises a CDR1α chain comprising the aminoacid sequence of SEQ ID NO: 58, a CDR2α chain consisting of the aminoacid sequence of SEQ ID NO: 59, a CDR3α chain comprising the amino acidsequence of SEQ ID NO: 60, a CDR1β chain comprising the amino acidsequence of SEQ ID NO: 66, a CDR2β chain consisting of the amino acidsequence of SEQ ID NO: 67, and a CDR3β chain comprising the amino acidsequence of SEQ ID NO:
 68. 19. The method of claim 1, wherein the TCRcomprises a CDR1α chain consisting of the amino acid sequence of SEQ IDNO: 58, a CDR2α chain comprising the amino acid sequence of SEQ ID NO:59, a CDR3α chain comprising the amino acid sequence of SEQ ID NO: 60, aCDR1β chain consisting of the amino acid sequence of SEQ ID NO: 66, aCDR2β chain comprising the amino acid sequence of SEQ ID NO: 67, and aCDR3β chain comprising the amino acid sequence of SEQ ID NO:
 68. 20. Themethod of claim 1, wherein the cancer is melanoma.